Assessing Cardiac Disease Via Detection and Measurement of MicroRNA

ABSTRACT

Disclosed are methods and materials for assessing cardiac disease, including cardiac failure, cardiac hypertrophy, thoracic aortic aneurysm, left ventricular remodeling using microRNA levels. The level of microRNAs can be measured in a body fluid, such as plasma and serum, or in cardiac tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. application Ser. No. 12/944,670,filed Nov. 11, 2010. Application Ser. No. 12/944,670, filed Nov. 11,2010, is hereby incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos.HL87134, HL81692, HL85531, RR16434, RR16461, HL057952-08, HL059165-09,and HL089170-01A1 awarded by the National Institutes of Health (NIH),and VA Merit Nos. 101 BX000904-01 and 101 CX000415-01A1 awarded by theDepartment of Veterans Affairs (VA). The government has certain rightsin the invention.

FIELD OF THE INVENTION

The disclosed invention is generally in the field of cardiac disease andcardiac failure and specifically in the area of diagnosis, prognosis,and monitoring of cardiac disease and cardiac failure.

BACKGROUND OF THE INVENTION

MicroRNAs (miRNAs) are noncoding RNAs that bind to target mRNAs andreduce their expression through translational repression or mRNAdegradation. Measurements made in myocardial tissue have suggested themiRNAs play a regulatory role in myocardial growth, fibrosis, andremodeling.

MicroRNAs have been isolated from C. elegans, Drosophila, and humans(Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001).Several hundred miRNAs have been identified in plants andanimals—including humans—which do not appear to have endogenous siRNAs.Thus, while similar to siRNAs, miRNAs are nonetheless distinct. miRNAsthus far observed are approximately 21-22 nucleotides in length and theyarise from longer precursors, which are transcribed fromnon-protein-encoding genes. See review of Carrington et al. (2003). Theprecursors form structures that fold back on each other inself-complementary regions; they are then processed by the nucleaseDicer in animals or DCL1 in plants. miRNA molecules interrupttranslation through imprecise base-pairing with their targets.

Most miRNAs are involved in gene regulation. Some of these miRNAs,including lin-4 and let-7, inhibit protein synthesis by binding topartially complementary 3′ untranslated regions (3′ UTRs) of targetmRNAs. Others, including the Scarecrow miRNA found in plants, functionlike siRNA and bind to perfectly complementary mRNA sequences to destroythe target transcript (Grishok et al., 2001). Some miRNAs, such aslin-4, let-7, mir-14, mir-23, and bantam, have been shown to playcritical roles in cell differentiation and tissue development (Ambros,2003; Xu et al., 2003). Others are believed to have similarly importantroles because of their differential spatial and temporal expressionpatterns.

BRIEF SUMMARY OF THE INVENTION

Disclosed are methods and materials for assessing cardiac disease,including cardiac failure, cardiac hypertrophy, thoracic aorticaneurysm, left ventricular remodeling using microRNA levels. The levelof microRNAs can be measured in a body fluid, such as plasma and serum,or in tissue, such as cardiac and aortic tissue.

Disclosed are methods comprising detecting one or more target microRNAsin tissue or a body fluid of a subject, where the level or amount of theone or more target microRNAs indicates, for example, the risk,development, presence, severity, or a combination, of thoracic aorticaneurysm in the subject, of diastolic heart failure in the subject, ofleft ventricular hypertrophy in the absence of diastolic heart failurein the subject, of left ventricular remodeling in the subject, that thesubject experienced ischemia-reperfusion, or a combination.

MicroRNAs can be used to assess or diagnose aortic aneurysm. Forexample, a level or amount in a subject of miR-133a, miR-21, miR-29a,miR-208, or a combination, lower than the level or amount in control,reference, and/or normal subjects and/or in a control or reference RNAin the subject or in control, reference, and/or normal subjectsindicates aortic aneurysm. The control or reference level can be, forexample, the level in normal aorta.

In some forms the methods can indicate the risk, presence, severity, ora combination, of thoracic aortic aneurysm in the subject. In someforms, the one or more target microRNAs can comprise miR-1, miR-21,miR-29a, miR-133a, and miR-486-5p, or a combination, wherein a level oramount of miR-1, miR-21, miR-29a, miR-133a, and miR-486-5p, or acombination, less than the level or amount in control subjects,reference subjects, normal subjects, or a combination, indicates therisk, presence, severity, or a combination, of thoracic aortic aneurysmin the subject.

In some forms, a set of microRNAs is detected and if a thresholdpercentage or more of the set of microRNAs has an indicated level oramount, the subject can be assessed for a disease or condition. Forexample, if a threshold percentage or more of a set of microRNAs has alevel or amount of less than or greater than (depending on theindividual microRNA) than the level or amount in control subjects,reference subjects, normal subjects, or a combination, it indicates arisk, presence, severity, or a combination, of thoracic aortic aneurysmin a subject.

In some forms, the one or more target microRNAs comprises miR-455-3p,miR-1268, miR-338-5p, miR-940, miR-1323, miR-768-3p, miR-574-3p,miR-106b, miR-451, miR-100, miR-125b, miR-195, miR-19b, miR-30d,miR-15b, miR-125a-5p, miR-143, miR-193b, miR-16, miR-27a, miR-29a,miR-30a, miR-27b, miR-92a, miR-140-5p, let-7i, miR-151-5p, miR-140-3p,miR-24, miR-23a, miR-145, miR-199b-3p, miR-199a-3p, miR-361-5p,miR-130a, miR-22, and miR-497. For miR-455-3p, miR-1268, miR-338-5p, andmiR-940, a level or amount greater than the level or amount in controlsubjects, reference subjects, normal subjects, or a combination, iscorrelated with the risk, presence, severity, or a combination, ofthoracic aortic aneurysm in the subject. For miR-1323, miR-768-3p,miR-574-3p, miR-106b, miR-451, miR-100, miR-125b, miR-195, miR-19b,miR-30d, miR-15b, miR-125a-5p, miR-143, miR-193b, miR-16, miR-27a,miR-29a, miR-30a, miR-27b, miR-92a, miR-140-5p, let-7i, miR-151-5p,miR-140-3p, miR-24, miR-23a, miR-145, miR-199b-3p, miR-199a-3p,miR-361-5p, miR-130a, miR-22, and miR-497, a level or amount less thanthe level or amount in control subjects, reference subjects, normalsubjects, or a combination, is correlated with the risk, presence,severity, or a combination, of thoracic aortic aneurysm in the subject.

In some forms, the threshold percentage can be on or about 60%, 65%,70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more of the target microRNAs.

In some forms, the one or more target microRNAs can comprise miR-1,miR-21, miR-29a, and miR-133a, or a combination, wherein the lower thelevel or amount of miR-1, miR-21, miR-29a, miR-133a, and miR-486-5p, ora combination, less than the level or amount in control subjects,reference subjects, normal subjects, or a combination, the larger thethoracic aortic aneurysm in the subject.

In some forms, the lower the level or amount of miR-29a less than thelevel or amount in control subjects, reference subjects, normalsubjects, or a combination, the higher the level of MMP-2 in thesubject. In some forms, the lower the level or amount of miR-133a lessthan the level or amount in control subjects, reference subjects, normalsubjects, or a combination, the higher the level of MMP-9 in thesubject. In some forms, prior to detection, the subject can be suspectedof having thoracic aortic aneurysm.

In some forms, decreased level (compared to normal) of miR-1, miR-21,miR-29a, miR-133a, and miR-486-5p is correlated with TAA (decreaseindicates TAA). In some forms, the magnitude of decreased level(compared to normal) of miR-1, miR-21, miR-29a, and miR-133a iscorrelated with size of TAA (larger decrease indicates larger TAA). Insome forms, the level (compared to normal) of miR-29a is inverselycorrelated with MMP-2 (decreased level of miR-29a indicates increasedlevel of MMP-2). In some forms, the level (compared to normal) ofmiR-133a is inversely correlated with MMP-9 (decreased level of miR-133aindicates increased level of MMP-9). In some forms, a decreased level of33 specific miRNAs and/or an increased level of 4 specific miRNAs arecorrelated with TAA (decrease indicates TAA).

In some forms the methods can indicate the risk, development, presence,severity, or a combination, of diastolic heart failure in a subjecthaving left ventricular hypertrophy. In some forms, the one or moretarget microRNAs can comprise miR-1, miR-21, miR-29a, miR-133a, andmiR-760, or a combination, wherein a decrease in the level or amount ofmiR-1, miR-21, miR-29a, miR-133a, and miR-760, or a combination, from alevel or amount greater than the level or amount in control subjects,reference subjects, normal subjects, or a combination, indicates therisk, development, presence, severity, or a combination, of diastolicheart failure in the subject.

In some forms, the one or more target microRNAs comprises miR-1, miR-21,miR-29a, miR-133a, and miR-760, or a combination, wherein a decrease inthe level or amount of miR-1, miR-21, miR-29a, miR-133a, and miR-760, ora combination, from a level or amount in subjects with left ventricularhypertrophy but not diastolic heart failure, indicates the risk,development, presence, severity, or a combination, of diastolic heartfailure in the subject.

The decrease in the level or amount of the one or more target microRNAscan be determined by detecting the level or amount of the one or moretarget microRNAs at different times. The level or amount of the one ormore target microRNAs at one or more of the different times can begreater than the level or amount in control subjects, referencesubjects, normal subjects, or a combination.

In some forms the methods can indicate the presence or absence ofdiastolic heart failure in a subject having left ventricularhypertrophy. In some forms, the one or more target microRNAs cancomprise miR-1, miR-21, miR-29a, miR-133a, and miR-760, or acombination, wherein a level or amount of miR-1, miR-21, miR-29a,miR-133a, and miR-760, or a combination, about the same as the level oramount in control subjects, reference subjects, normal subjects, or acombination, indicates the presence of diastolic heart failure in thesubject.

In some forms, the one or more target microRNAs comprises miR-1, miR-21,miR-29a, miR-133a, and miR-760, or a combination, wherein a level oramount of miR-1, miR-21, miR-29a, miR-133a, and miR-760, or acombination, greater than the level or amount in control subjects,reference subjects, normal subjects, or a combination, indicates theabsence of diastolic heart failure in the subject.

In some forms, increased level (compared to normal) of miR-1, miR-21,miR-29a, miR-133a, and miR-760 is correlated with LVH without DHF(increase indicates LVH without DHF). In some forms, unchanged level(compared to normal) of miR-1, miR-21, miR-29a, miR-133a, and miR-760 iscorrelated with LVH with DHF (normal in LVH patients indicates LVH withDHF). In some forms, change in level from increased (compared to normal)of miR-1, miR-21, miR-29a, miR-133a, and miR-760 is correlated withdevelopment of DHF in LVH patients (return to normal in LVH patientsindicates LVH with DHF).

In some forms the methods can indicate that the subject experiencedischemia-reperfusion. In some forms, the one or more target microRNAscan comprise miR-1, miR-133a, miR-760, or a combination, wherein a levelor amount of miR-1, miR-133a, miR-760, or a combination, greater thanthe level or amount in control subjects, reference subjects, normalsubjects, or a combination, indicates that the subject experiencedischemia-reperfusion. In some forms, the subject can be suspected ofhaving experienced ischemia-reperfusion. In some forms, increased level(compared to normal) of miR-1, miR-133a, and miR-760 is correlated withischemia-reperfusion (increase indicates I/R).

As another example, a level or amount in a subject of miR-1, miR-208,miR-133a, miR-21, miR-29a, or a combination, higher than the level oramount in control, reference, and/or normal subjects and/or in a controlor reference RNA in the subject or in control, reference, and/or normalsubjects followed by a level or amount in the subject of miR-1, miR-21,miR-29a, miR-133a, miR-760, or a combination, lower than the level oramount of miR-1, miR-21, miR-29a, miR-133a, miR-760, respectively,indicates development of diastolic heart failure. As another example, ina subject with left ventricular remodeling but not diastolic heartfailure, a level or amount in the subject of miR-1, miR-21, miR-29a,miR-133a, miR-760, or a combination, lower than the level or amount ofmiR-1, miR-21, miR-29a, miR-133a, miR-760, respectively, measuredearlier in the subject indicates development of diastolic heart failure.

In some forms the methods can indicate a higher risk of left ventricularremodeling in the subject. In some forms, the one or more targetmicroRNAs can be detected following myocardial infarction in thesubject.

In some forms, the one or more target microRNAs can comprise miR-1,miR-21, or a combination, wherein the one or more target microRNAs aredetected on or about 2 days following myocardial infarction, wherein alevel or amount of miR-1, miR-21, or a combination, less than the levelor amount in control subjects, reference subjects, normal subjects, or acombination, indicates a higher risk of left ventricular remodeling inthe subject.

In some forms the methods can indicate a higher risk of increased leftventricular end diastolic volume developing in the subject. In someforms, the one or more target microRNAs can comprise miR-29a, whereinthe one or more target microRNAs are detected on, about, or between 4and 10 days following myocardial infarction, wherein a level or amountof miR-29a more than the level or amount in control subjects, referencesubjects, normal subjects, or a combination, indicates a higher risk ofincreased left ventricular end diastolic volume developing in thesubject.

In some forms, the one or more target microRNAs can be detected on,about, or between 5 and 8 days following myocardial infarction. In someforms, the one or more target microRNAs can be detected on or about 5days following myocardial infarction.

In some forms, miR-1 and miR-21 levels decreased (compared to normal) at2 days post-MI correlated with LVR. In some forms, miR-29a levels(compared to normal) early after MI (e.g., day 5) is correlated withleft ventricular end diastolic volume (LV EDV) late after MI (e.g., day90).

In some forms, the one or more target microRNAs are detected in atissue. The tissue can be cardiac tissue. The tissue can be thoracicaortic tissue. In some forms, the one or more target microRNAs aredetected in a body fluid. The body fluid can be plasma. In some forms,the one or more target microRNAs can be detected in a tissue sample or abody fluid sample collected from the subject. In some forms, detectionof the one or more microRNAs can be accomplished by altering the tissueor body fluid so that the microRNAs produce a signal. In some forms, thealteration of the tissue or body sample comprising amplifying themicroRNAs. For example, the microRNAs can be amplified via polymerasechain reaction (PCR). In some forms, the alteration can be hybridizationof a probe to the microRNAs or amplified copies of the microRNAsequences.

In some forms, the level of the one or more target microRNAs cancomprise the measured level of the one or more target microRNAsnormalized to the measured level of a reference RNA in the tissue orbody fluid. In some forms, the reference RNA can be snRNA U6.

In some forms, the level of the one or more target microRNAs cancomprise the measured level of the one or more target microRNAsexpressed as the fold difference of the measured level of the one ormore target microRNAs to the measured level of the one or more targetmicroRNAs in a reference subject. In some forms, the level of the one ormore target microRNAs can comprise the measured level of the one or moretarget microRNAs normalized to the measured level of a reference RNA inthe tissue or body fluid expressed as the fold difference of thenormalized level of the one or more target microRNAs to the measuredlevel of the one or more target microRNAs in the same tissue or bodyfluid of reference subject normalized to the measured level of areference RNA in the tissue or body fluid of the reference subject.

In some forms, the level of the one or more target microRNAs in areference subject can be measured at the same time as the level of theone or more target microRNAs is measured in the subject. In some forms,the level of the one or more target microRNAs in a reference subject canbe measured at a different time than the level of the one or more targetmicroRNAs is measured in the subject. In some forms, the level of theone or more target microRNAs in a reference subject can be a referencelevel.

In some forms, the method can further comprise treating the subject forthoracic aortic aneurysm. In some forms, the method can further comprisetreating the subject for diastolic heart failure. In some forms, themethod can further comprise treating the subject forischemia-reperfusion injury. In some forms, the method can furthercomprise treating the subject for left ventricular remodeling.

Also disclosed are methods comprising detecting one or more targetmicroRNAs in a body fluid of a subject at a plurality of differenttimes. The temporal pattern of the level of the one or more targetmicroRNAs can indicates the presence, severity, or a combination of leftventricular remodeling in the subject.

The presence, severity, or a combination of left ventricular remodelingin the subject can be indicated by comparing the temporal pattern of thelevel of the one or more target microRNAs to one or more referencetemporal patterns. The one or more microRNAs can comprise one or more ofmiR-1, miR-21, miR-23a, miR-29a, miR-30, miR-133a, miR-150, miR-195,miR-199, miR-208, miR-214, and miR-125b. The one or more microRNAs cancomprise one or more of miR-1, miR-21, miR-29a, miR-133a, miR-208, andmiR-125b. The one or more microRNAs can comprise one or more of miR-1,miR-21, miR-29a, miR-133a, and miR-208.

The body fluid can be, for example, blood, plasma, serum, or lymphaticfluid. The plurality of different times at which the one or moremicroRNAs are detected can comprise two or more times separated by 1, 2,3, 4, 5, 10, 15, 20, 23, 24, 25, 26, 27, 28, 30, 35, 40, 45, 50, 55, 60,62, 65, 70, 75, 80, 85, 86, 87, 88, 89, and 90 days. The plurality ofdifferent times at which the one or more microRNAs are detected cancomprise two or more times separated by 2, 3, 23, and 62 days. The levelof the one or more target microRNAs can comprise the measured level ofthe one or more target microRNAs normalized to the measured level of areference RNA in the body fluid. The reference RNA can be snRNA U6.

The level of the one or more target microRNAs can comprise the measuredlevel of the one or more target microRNAs expressed as the folddifference of the measured level of the one or more target microRNAs tothe measured level of the one or more target microRNAs in a referencesubject. The level of the one or more target microRNAs can comprise themeasured level of the one or more target microRNAs normalized to themeasured level of a reference RNA in the body fluid expressed as thefold difference of the normalized level of the one or more targetmicroRNAs to the measured level of the one or more target microRNAs inthe same body fluid of reference subject normalized to the measuredlevel of a reference RNA in the body fluid of the reference subject.

The level of the one or more target microRNAs in a reference subject canbe measured at the same time as the level of the one or more targetmicroRNAs is measured in the subject. The level of the one or moretarget microRNAs in a reference subject can be measured at a differenttime than the level of the one or more target microRNAs is measured inthe subject. The level of the one or more target microRNAs in areference subject can be a reference level.

The plurality of different times can comprise two or more times 1, 2, 3,4, 5, 10, 15, 20, 25, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, and 90 days following a known or suspected myocardial infarction inthe subject. The plurality of different times can comprise two or moretimes 2, 5, 28, and 90 days following a known or suspected myocardialinfarction in the subject. The temporal pattern of the level of the oneor more target microRNAs can indicate that the subject suffered amyocardial infarction. The temporal pattern of the level of the one ormore target microRNAs can indicate how long ago the subject suffered themyocardial infarction.

Additional advantages of the disclosed method and compositions will beset forth in part in the description which follows, and in part will beunderstood from the description, or may be learned by practice of thedisclosed method and compositions. The advantages of the disclosedmethod and compositions will be realized and attained by means of theelements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of thedisclosed method and compositions and together with the description,serve to explain the principles of the disclosed method andcompositions.

FIG. 1 is a diagram showing production and function of microRNA.

FIG. 2 is a chart showing micro RNAs involved in heart disease.

FIG. 3 is a graph of end diastolic volume (EDV) versus days aftermyocardial infarction. EDV increases while EDV of age and gender matchedreferent controls does not.

FIG. 4 is a graph of the fold change of miR-1 (compared to age matchednormals) versus days following myocardial infarction.

FIG. 5 is a graph of the fold change of miR-133a (compared to agematched normals) versus days following myocardial infarction.

FIG. 6 is a graph of the fold change of miR-208 (compared to age matchednormals) versus days following myocardial infarction.

FIG. 7 is a graph of the fold change of miR-21 (compared to age matchednormals) versus days following myocardial infarction.

FIG. 8 is a graph of the fold change of miR-29a (compared to age matchednormals) versus days following myocardial infarction.

FIG. 9 is a graph of the fold change of microRNA-29a (compared to agematched normals) at 5 days after myocardial infarction versus leftventricular end diastolic volume at 90 days after myocardial infarction.The level of microRMA-29a at 5 days after myocardial infarction ispositively correlated to left ventricular end diastolic volume at 90days after myocardial infarction.

FIG. 10 is a graph of quantitative PCR (QPCR) production versus cyclenumber for various serial dilutions of an miRNA standard.

FIG. 11 is a graph of quantitative PCR production of snRNA U6 frompatients 5 days after myocardial infarction and age matched normals.Lower panel is an enlargement of the boxed area in the upper panel.

FIG. 12 is a graph of quantitative PCR production of microRNA-1 frompatients 5 days after myocardial infarction and age matched normals.Lower panel is an enlargement of the boxed area in the upper panel.

FIG. 13 is a table showing the coefficient of variation in miRNAquantifications for various microRNAs. Also shown is a graph of thecorrelation line for miR-21.

FIG. 14 is a graph of quantitative PCR production of miR-1 versus cyclenumber for various microRNA samples.

FIG. 15 is a graph of quantitative PCR production of miR-21 versus cyclenumber for various microRNA samples.

FIG. 16 is a graph of quantitative PCR production of miR-29 versus cyclenumber for various microRNA samples.

FIG. 17 is a graph of quantitative PCR production of miR-125b versuscycle number for various microRNA samples.

FIG. 18 is a graph of quantitative PCR production of miR-133 versuscycle number for various microRNA samples.

FIG. 19 is a graph of quantitative PCR production of miR-208 versuscycle number for various microRNA samples.

FIG. 20 shows an increase of miRs in patients with LVH compared to DHF.

FIG. 21 is a graph of the relative expression levels of miRs determinedby quantitative real-time PCR.

FIG. 22 shows a comparison of miRs in INTf and Plasma as well as thechange following I/R.

FIG. 23 is a graph showing that high frequency electrical stimulation ofhuman myocardial fibroblasts causes differential expression ofmicroRNAs.

FIG. 24 shows that the end diastolic volume increased progressivelyfollowing a myocardial infarction (closed circles) compared withreferent controls (shaded box=normal range). *p<0.05 vs. controls.

FIGS. 25A, 25B, 25C and 25D are examples of the quantitative real timePolymerase Chain Reaction (Qrt-PCR) in a referent control (CTL) versus apatient 5 days following a myocardial infarction (post-MI). snRNA U6(panel A and B) expression (measured by cycle time) was not changed 5days post MI. miR-1 (panel C and D) expression was increased 5 days postMI as evident by the reduction in cycle time from 30.7 in CTL to 28.1for post-MI (arrow).

FIG. 26 shows serial changes in miRs following a myocardial infarction(MI). Post-MI data are presented as a fold change from controls (CTL)set at 1. *=P<0.05 vs. CTL; #=P<0.05 vs. day 2.

FIGS. 27A and 27B show changes in miR-29. Panel A shows serial changesin miR-29 (fold change from referent controls set at 1) following amyocardial infarction (MI). *=P<0.05 vs. CTL; #=P<0.05 vs. day 2. PanelB shows the relationship between early changes in miR-29a 5 days post-MIversus late changes in end diastolic volume 90 days post-MI. The largerthe early increase in miR-29a at 5 days, the larger the late increase LVend diastolic volume following an MI. y=−5.5+0.07x, r=0.77.

FIG. 28 shows the alterations in miR expression in clinical TAAspecimens as compared to normal aorta. Mean percent change (±SEM) of miRexpression in clinical TAA specimens versus normal aorta. All specimenswere analyzed by quantitative PCR. The results for all cycling specimensare shown in the graph. The dashed line represents the results fornormal aorta set at 100% for each miR examined (n=minimum of 8). Thebars represent results for miR expression in cycling TAA specimens;miR-1 (n=21), miR-21 (n=26), miR-29a (n−27), miR-133a (n=25), miR-486-5p(n=26), and miR-760 (n=26); * indicates p<0.05 versus 100%. Note:expression of the myocardial-specific miR-208 was not detected in ouraortic specimens.

FIGS. 29A and 29B show differentially expressed miRs in clinical TAAspecimens as compared to normal aorta. miRs differentially expressedbetween aneurysmal (Aneur; n=4) and normal (Normal, n=4) aorta weredetected by Affymetrix GeneChip analysis. A, Heatmap depicting 37differentially expresses miRs between Aneur and Normal aorta.Colorimetric scaling for expression values (z-standardized) is shown atbottom. Patterns for miR-29a, and previously describe miR-143/miR-145,are indicated. B, Schematic diagram depicting detection scoring for 106miRs that changed from detected to undetected or undetected to detectedbetween Aneur and Normal aorta. Positive statistical detection calls formiRs are indicated by blue; absent (i.e., undetected) calls areindicated by yellow. Patterns for miRs-1, -133a, and -486-5p areindicated.

FIGS. 30A and 30B show miR expression and relationship to aortic size.A. miR expression was stratified into groups based on aortic diameter(TAA size) defined as: small TAAs (4.0-5.0 cm), medium TAAs (5.1-6.0cm), or large TAAs (6.1-7.5 cm), and results were compared to normalaorta (Normal). Expression levels were calculated as a percent changeform normal aorta (set at 100%) and displayed as box plots showing themedian (solid line), interquartile range (25^(th) to 75^(th) percentile;gray box), and the mean (dashed line), overlaid with a scatter plot ofeach value; *p<0.05 versus 100%, #p<0.05 versus small TAAs. B. Linearleast-squares regression analysis demonstrating several significantinverse relationships between miR expression in clinical TAA specimensand aortic diameter; miR-1 (r=−0.5433, p=0.0109, n=21), miR-21(r=−0.4132, p=0.0359, n=26), miR-29a (r=−0.5364, p=0.0039, n=27), andmiR-133a (r=−0.4247, p=0.0344, n=25).

FIGS. 31A, 31B, and 31C show the identification and relative abundanceof potential biological targets for altered miRs in clinical TAAspecimens. A. Significant miR binding sites identified using theTargetScanHuman database (v5.1 released April 2009) (SEQ ID NOs:7, 3, 8,4). B. Relative protein abundance of MMP-2 in clinical TAA specimens ascompared to normal aorta as determined by zymographic analysis. C.Relative protein abundance of MMP-9 in clinical TAA specimens ascompared to normal aorta as determined by zymographic analysis. Changesin the ratio of active:total forms were determined by densitometry andexpressed as mean percent change (±SEM) from normal aorta (set at 100%);*p<0.05 versus 100%.

FIG. 32 shows the modulation of miR-29a expression levels in humanprimary aortic vascular smooth muscle cells. Cells were exposed to thetransduction vehicle alone, miR-29a lentivirus, or anti-miR-29alentivirus. Five days post-transduction cells were harvested andexamined by gelatin zymography. The results demonstrated thatoverexpression of miR-29a attenuated total MMP-2 protein abundance,while overexpression of anti-miR-29a enhanced total MMP-2 proteinabundance (n=3; *p<0.05 versus control, #p<0.05 versus miR-29atransduced cells).

FIG. 33 shows the relationship between total MMP-2 abundance and miR-29aexpression in clinical TAA specimens. Linear least-squares regressionanalysis demonstrated a significant inverse relationship between miR-29aexpression and total MMP-2 abundance (r=−0.4198, p=0.0209, n=30).

FIG. 34 shows the alterations in miR expression in clinical TAAspecimens as compared to normal aorta. Representative plots of rawcycling data depicting the difference in ΔCt values on the x-axis andthe change in relative fluorescence values on the y-axis, between normalaorta (solid line) and TAA aorta (dashed line) from quantitativereal-time PCR results.

FIG. 35 shows the functional characteristics of the 37 differentiallyexpressed miRs between Aneurysm (n=4) and Normal (n=4) aorta. Thefunctions for the 37 differentially expressed miRs were estimated basedon published manuscripts reported in PubMed.; 27% target proliferationpathways, 16% target growth arrest pathways, 8% target ECMstructure/function, 5% (each) target apoptosis, migration, andangiogenesis, 3% (each) target drug resistance, immune response,autophagy, and cell adhesion pathways, and 22% to date have no reportedfunction.

FIG. 36 shows the transduction of human primary aortic vascular smoothmuscle cells with a non-targeting mismatch control virus. Quantitationof total MMP-2 protein abundance following viral transduction. Theresults demonstrated that overexpression of a non-targeting sequence hadno effect on total MMP-2 protein levels (n=3; p=0.6047).

DETAILED DESCRIPTION OF THE INVENTION

The disclosed method and compositions may be understood more readily byreference to the following detailed description of particularembodiments and the Example included therein and to the Figures andtheir previous and following description.

Disclosed are methods and materials for assessing cardiac disease,including cardiac failure, cardiac hypertrophy, thoracic aorticaneurysm, left ventricular remodeling using microRNA levels. The levelof microRNAs can be measured in a body fluid, such as plasma and serum,or in tissue, such as cardiac and aortic tissue. For example, disclosedare methods comprising detecting one or more target microRNAs in tissueor a body fluid of a subject, where the level or amount of the one ormore target microRNAs indicates, for example, the risk, development,presence, severity, or a combination, of thoracic aortic aneurysm in thesubject, of diastolic heart failure in the subject, of left ventricularhypertrophy in the absence of diastolic heart failure in the subject, ofleft ventricular remodeling in the subject, that the subject experiencedischemia-reperfusion, or a combination.

“Detecting” or like terms refers to measuring or otherwise determiningan object or magnitude to be detected. For example, detection can referto determining the presence or absence of an object or to determining ormeasuring a magnitude, such as a level, amount, or size of an object.“Level” and “amount” of an object can refer to, for example, a number ormass of the object, such as a dozen eggs or 100 mg of a molecule or to aconcentration, such a molarity or grams per liter of the object or amolecule.

MicroRNAs can be used to assess or diagnose aortic aneurysm. Forexample, in some forms the methods can indicate the risk, presence,severity, or a combination, of thoracic aortic aneurysm in the subject.

In some forms the methods can indicate the risk, presence, severity, ora combination, of thoracic aortic aneurysm in the subject. In someforms, the one or more target microRNAs can comprise miR-1, miR-21,miR-29a, miR-133a, and miR-486-5p, or a combination, wherein a level oramount of miR-1, miR-21, miR-29a, miR-133a, and miR-486-5p, or acombination, less than the level or amount in control subjects,reference subjects, normal subjects, or a combination, indicates therisk, presence, severity, or a combination, of thoracic aortic aneurysmin the subject.

Reference herein to level(s) or amount(s) in control subjects, referencesubjects, normal subjects, or a combination should be understood toinclude the use of level(s) or amount(s) in one control subject,multiple control subjects, one reference subject, multiple referencesubjects, one normal subject, multiple normal subjects, or anycombination of these. As used herein, a normal subject refers to asubject that does not have the relevant condition or disease. As usedherein, reference subject refers to a subject that serves as a referencefor the measurement to be made. For example, a reference subject can bea normal subject (when comparing for alterations or abnormal levels oramounts) or an affected subject (when comparison to one form or type ofthe disease or condition is indicated). As used herein, control subjectrefers to a subject that serves as a control for test or assessedsubjects. For example, a control subject can be a normal subject or areference subject.

As used herein, a reference level refers to a level or amount thatserves as a reference for the measurement to be made. For example, areference level can be a normal level or amount (from a normal subject,normal tissue and/or normal body fluid), a control level or amount(from, for example, a normal subject, normal tissue and/or normal bodyfluid, or an affected subject, affected tissue and/or affected bodyfluid)

In some forms, a set of microRNAs is detected and if a thresholdpercentage or more of the set of microRNAs has an indicated level oramount, the subject can be assessed for a disease or condition. Forexample, if a threshold percentage or more of a set of microRNAs has alevel or amount of less than or greater than (depending on theindividual microRNA) than the level or amount in control subjects,reference subjects, normal subjects, or a combination, it indicates arisk, presence, severity, or a combination, of thoracic aortic aneurysmin a subject.

In some forms, the one or more target microRNAs comprises miR-455-3p,miR-1268, miR-338-5p, miR-940, miR-1323, miR-768-3p, miR-574-3p,miR-106b, miR-451, miR-100, miR-125b, miR-195, miR-19b, miR-30d,miR-15b, miR-125a-5p, miR-143, miR-193b, miR-16, miR-27a, miR-29a,miR-30a, miR-27b, miR-92a, miR-140-5p, let-7i, miR-151-5p, miR-140-3p,miR-24, miR-23a, miR-145, miR-199b-3p, miR-199a-3p, miR-361-5p,miR-130a, miR-22, and miR-497. For miR-455-3p, miR-1268, miR-338-5p, andmiR-940, a level or amount greater than the level or amount in controlsubjects, reference subjects, normal subjects, or a combination, iscorrelated with the risk, presence, severity, or a combination, ofthoracic aortic aneurysm in the subject. For miR-1323, miR-768-3p,miR-574-3p, miR-106b, miR-451, miR-100, miR-125b, miR-195, miR-19b,miR-30d, miR-15b, miR-125a-5p, miR-143, miR-193b, miR-16, miR-27a,miR-29a, miR-30a, miR-27b, miR-92a, miR-140-5p, let-7i, miR-151-5p,miR-140-3p, miR-24, miR-23a, miR-145, miR-199b-3p, miR-199a-3p,miR-361-5p, miR-130a, miR-22, and miR-497, a level or amount less thanthe level or amount in control subjects, reference subjects, normalsubjects, or a combination, is correlated with the risk, presence,severity, or a combination, of thoracic aortic aneurysm in the subject.

In some forms, the threshold percentage can be on or about 60%, 65%,70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more of the target microRNAs.

In some forms, the one or more target microRNAs can comprise miR-1,miR-21, miR-29a, and miR-133a, or a combination, wherein the lower thelevel or amount of miR-1, miR-21, miR-29a, miR-133a, and miR-486-5p, ora combination, less than the level or amount in control subjects,reference subjects, normal subjects, or a combination, the larger thethoracic aortic aneurysm in the subject.

In some forms, the lower the level or amount of miR-29a less than thelevel or amount in control subjects, reference subjects, normalsubjects, or a combination, the higher the level of MMP-2 in thesubject. In some forms, the lower the level or amount of miR-133a lessthan the level or amount in control subjects, reference subjects, normalsubjects, or a combination, the higher the level of MMP-9 in thesubject. In some forms, prior to detection, the subject can be suspectedof having thoracic aortic aneurysm.

MicroRNAs can be used to assess or diagnose diastolic heart failure. Forexample, in some forms the methods can indicate the risk, development,presence, severity, or a combination, of diastolic heart failure in asubject having left ventricular hypertrophy.

In some forms, the one or more target microRNAs can comprise miR-1,miR-21, miR-29a, miR-133a, and miR-760, or a combination, wherein adecrease in the level or amount of miR-1, miR-21, miR-29a, miR-133a, andmiR-760, or a combination, from a level or amount greater than the levelor amount in control subjects, reference subjects, normal subjects, or acombination, indicates the risk, development, presence, severity, or acombination, of diastolic heart failure in the subject.

In some forms, the one or more target microRNAs comprises miR-1, miR-21,miR-29a, miR-133a, and miR-760, or a combination, wherein a decrease inthe level or amount of miR-1, miR-21, miR-29a, miR-133a, and miR-760, ora combination, from the level or amount in subjects with leftventricular hypertrophy but not diastolic heart failure, indicates therisk, development, presence, severity, or a combination, of diastolicheart failure in the subject.

The decrease in the level or amount of the one or more target microRNAscan be determined by detecting the level or amount of the one or moretarget microRNAs at different times. The level or amount of the one ormore target microRNAs at one or more of the different times can begreater than the level or amount in control subjects, referencesubjects, normal subjects, or a combination.

In some forms the methods can indicate the presence or absence ofdiastolic heart failure in a subject having left ventricularhypertrophy. In some forms, the one or more target microRNAs cancomprise miR-1, miR-21, miR-29a, miR-133a, and miR-760, or acombination, wherein a level or amount of miR-1, miR-21, miR-29a,miR-133a, and miR-760, or a combination, about the same as the level oramount in control subjects, reference subjects, normal subjects, or acombination, indicates the presence of diastolic heart failure in thesubject.

In some forms, the one or more target microRNAs comprises miR-1, miR-21,miR-29a, miR-133a, and miR-760, or a combination, wherein a level oramount of miR-1, miR-21, miR-29a, miR-133a, and miR-760, or acombination, greater than the level or amount in control subjects,reference subjects, normal subjects, or a combination, indicates theabsence of diastolic heart failure in the subject.

MicroRNAs can be used to assess or diagnose ischemia-reperfusion. Forexample, in some forms the methods can indicate that the subjectexperienced ischemia-reperfusion. In some forms, the one or more targetmicroRNAs can comprise miR-1, miR-133a, miR-760, or a combination,wherein a level or amount of miR-1, miR-133a, miR-760, or a combination,greater than the level or amount in control subjects, referencesubjects, normal subjects, or a combination, indicates that the subjectexperienced ischemia-reperfusion. In some forms, the subject can besuspected of having experienced ischemia-reperfusion.

MicroRNAs can be used to assess or diagnose left ventricular remodelingfollowing myocardial infarction in the subject. For example, in someforms the methods can indicate a higher risk of left ventricularremodeling in the subject. In some forms, the one or more targetmicroRNAs can be detected following myocardial infarction.

In some forms, the one or more target microRNAs can comprise miR-1,miR-21, or a combination, wherein the one or more target microRNAs aredetected on or about 2 days following myocardial infarction, wherein alevel or amount of miR-1, miR-21, or a combination, less than the levelor amount in control subjects, reference subjects, normal subjects, or acombination, indicates a higher risk of left ventricular remodeling inthe subject.

In some forms the methods can indicate a higher risk of increased leftventricular end diastolic volume developing in the subject. In someforms, the one or more target microRNAs can comprise miR-29a, whereinthe one or more target microRNAs are detected on, about, or between 4and 10 days following myocardial infarction, wherein a level or amountof miR-29a more than the level or amount in control subjects, referencesubjects, normal subjects, or a combination, indicates a higher risk ofincreased left ventricular end diastolic volume developing in thesubject.

In some forms, the one or more target microRNAs can be detected on,about, or between 5 and 8 days following myocardial infarction. In someforms, the one or more target microRNAs can be detected on or about 5days following myocardial infarction.

In some forms, the one or more target microRNAs are detected in atissue. The tissue can be cardiac tissue. The tissue can be thoracicaortic tissue. In some forms, the one or more target microRNAs aredetected in a body fluid. The body fluid can be plasma. In some forms,the one or more target microRNAs can be detected in a tissue sample or abody fluid sample collected from the subject. In some forms, detectionof the one or more microRNAs can be accomplished by altering the tissueor body fluid so that the microRNAs produce a signal. In some forms, thealteration of the tissue or body sample comprising amplifying themicroRNAs. For example, the microRNAs can be amplified via polymerasechain reaction (PCR). In some forms, the alteration can be hybridizationof a probe to the microRNAs or amplified copies of the microRNAsequences.

“Collecting” or like terms refers to obtaining, extracting, removing, orotherwise separating something from a starting location. For example,collecting tissue, a body fluid, a sample, a tissue sample, a body fluidsample, etc. from a subject refers to obtaining, extracting, removing,or otherwise separating the tissue, body fluid, sample, tissue sample,body fluid sample, etc. from the subject. “Altering” or like termsrefers to transforming or combining an object. For example, alteringtissue, a body fluid, a sample, a tissue sample, a body fluid sample,microRNAs, etc. can refer to, for example, combining the tissue, bodyfluid, sample, tissue sample, body fluid sample, microRNA, etc. withcompositions that transform the tissue, body fluid, sample, tissuesample, body fluid sample, microRNAs, etc. or produce a signal from thetissue, body fluid, sample, tissue sample, body fluid sample, microRNAs,etc.

In some forms, the level of the one or more target microRNAs cancomprise the measured level of the one or more target microRNAsnormalized to the measured level of a reference RNA in the tissue orbody fluid. In some forms, the reference RNA can be snRNA U6.

In some forms, the level of the one or more target microRNAs cancomprise the measured level of the one or more target microRNAsexpressed as the fold difference of the measured level of the one ormore target microRNAs to the measured level of the one or more targetmicroRNAs in a reference subject. In some forms, the level of the one ormore target microRNAs can comprise the measured level of the one or moretarget microRNAs normalized to the measured level of a reference RNA inthe tissue or body fluid expressed as the fold difference of thenormalized level of the one or more target microRNAs to the measuredlevel of the one or more target microRNAs in the same tissue or bodyfluid of reference subject normalized to the measured level of areference RNA in the tissue or body fluid of the reference subject.

In some forms, the level of the one or more target microRNAs in areference subject can be measured at the same time as the level of theone or more target microRNAs is measured in the subject. In some forms,the level of the one or more target microRNAs in a reference subject canbe measured at a different time than the level of the one or more targetmicroRNAs is measured in the subject. In some forms, the level of theone or more target microRNAs in a reference subject can be a referencelevel.

In some forms, the method can further comprise treating the subject forthoracic aortic aneurysm. In some forms, the method can further comprisetreating the subject for diastolic heart failure. In some forms, themethod can further comprise treating the subject forischemia-reperfusion injury. In some forms, the method can furthercomprise treating the subject for left ventricular remodeling.

Also disclosed are methods and materials for assessing cardiac failure,cardiac hypertrophy, and left ventricular remodeling using microRNAlevels. The level of microRNAs can be measured in a body fluid, such asplasma and serum. Disclosed is method comprising detecting one or moretarget microRNAs in a body fluid of a subject at a plurality ofdifferent times. The temporal pattern of the level of the one or moretarget microRNAs can indicates the presence, severity, or a combinationof left ventricular remodeling in the subject.

The presence, severity, or a combination of left ventricular remodelingin the subject can be indicated by comparing the temporal pattern of thelevel of the one or more target microRNAs to one or more referencetemporal patterns. The one or more microRNAs can comprise one or more ofmiR-1, miR-21, miR-23a, miR-29a, miR-30, miR-133a, miR-150, miR-195,miR-199, miR-208, miR-214, and miR-125b. The one or more microRNAs cancomprise one or more of miR-1, miR-21, miR-29a, miR-133a, miR-208, andmiR-125b. The one or more microRNAs can comprise one or more of miR-1,miR-21, miR-29a, miR-133a, and miR-208.

The body fluid can be, for example, blood, plasma, serum, or lymphaticfluid. The plurality of different times at which the one or moremicroRNAs are detected can comprise two or more times separated by 1, 2,3, 4, 5, 10, 15, 20, 23, 24, 25, 26, 27, 28, 30, 35, 40, 45, 50, 55, 60,62, 65, 70, 75, 80, 85, 86, 87, 88, 89, and 90 days. The plurality ofdifferent times at which the one or more microRNAs are detected cancomprise two or more times separated by 2, 3, 23, and 62 days. The levelof the one or more target microRNAs can comprise the measured level ofthe one or more target microRNAs normalized to the measured level of areference RNA in the body fluid. The reference RNA can be snRNA U6.

The level of the one or more target microRNAs can comprise the measuredlevel of the one or more target microRNAs expressed as the folddifference of the measured level of the one or more target microRNAs tothe measured level of the one or more target microRNAs in a referencesubject. The level of the one or more target microRNAs can comprise themeasured level of the one or more target microRNAs normalized to themeasured level of a reference RNA in the body fluid expressed as thefold difference of the normalized level of the one or more targetmicroRNAs to the measured level of the one or more target microRNAs inthe same body fluid of reference subject normalized to the measuredlevel of a reference RNA in the body fluid of the reference subject.

The level of the one or more target microRNAs in a reference subject canbe measured at the same time as the level of the one or more targetmicroRNAs is measured in the subject. The level of the one or moretarget microRNAs in a reference subject can be measured at a differenttime than the level of the one or more target microRNAs is measured inthe subject. The level of the one or more target microRNAs in areference subject can be a reference level.

The plurality of different times can comprise two or more times 1, 2, 3,4, 5, 10, 15, 20, 25, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, and 90 days following a known or suspected myocardial infarction inthe subject. The plurality of different times can comprise two or moretimes 2, 5, 28, and 90 days following a known or suspected myocardialinfarction in the subject. The temporal pattern of the level of the oneor more target microRNAs can indicate that the subject suffered amyocardial infarction. The temporal pattern of the level of the one ormore target microRNAs can indicate how long ago the subject suffered themyocardial infarction.

MicroRNAs (miRNAs) are noncoding RNAs that bind to target mRNAs andreduce their expression through translational repression or mRNAdegradation. Measurements made in myocardial tissue have suggested themiRNAs play a regulatory role in myocardial growth, fibrosis, andremodeling. However, whether specific temporal changes in miRNAs occurin patients during the left ventricular (LV) remodeling process thatfollows myocardial infarction (MI) has not previously been demonstrated.

It has been discovered that miRNAs relevant to cardiac disease,including left ventricular remodeling, can be reliably measured in bodyfluids such as plasma and serum, that the levels of these miRNAs followtemporal patterns following myocardial infarction and during remodeling,and that these temporal patterns are highly correlated to the severityof left ventricular remodeling. Based on this, the disclosed methods forassessing cardiac failure, cardiac hypertrophy, and left ventricularremodeling using microRNA levels have been developed.

It is to be understood that the disclosed method and compositions arenot limited to specific synthetic methods, specific analyticaltechniques, or to particular reagents unless otherwise specified, and,as such, may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

Materials

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed method and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a microRNA measurement is disclosed anddiscussed and a number of modifications that can be made to the methodare discussed, each and every combination and permutation of themodifications that are possible are specifically contemplated unlessspecifically indicated to the contrary. Thus, if a class of molecules A,B, and C are disclosed as well as a class of molecules D, E, and F andan example of a combination molecule, A-D is disclosed, then even ifeach is not individually recited, each is individually and collectivelycontemplated. Thus, is this example, each of the combinations A-E, A-F,B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated andshould be considered disclosed from disclosure of A, B, and C; D, E, andF; and the example combination A-D. Likewise, any subset or combinationof these is also specifically contemplated and disclosed. Thus, forexample, the sub-group of A-E, B-F, and C-E are specificallycontemplated and should be considered disclosed from disclosure of A, B,and C; D, E, and F; and the example combination A-D. This conceptapplies to all aspects of this application including, but not limitedto, steps in methods of making and using the disclosed compositions.Thus, if there are a variety of additional steps that can be performedit is understood that each of these additional steps can be performedwith any specific embodiment or combination of embodiments of thedisclosed methods, and that each such combination is specificallycontemplated and should be considered disclosed.

A. Primers

Primers for use in the disclosed methods can be oligonucleotides havingsequence complementary to the target sequence. This sequence is referredto as the complementary portion of the primer. The complementary portionof a primer can be any length that supports specific and stablehybridization between the primer and the target sequence. Generally thisis 10 to 35 nucleotides long, but is preferably 16 to 24 nucleotideslong. For whole genome amplification, it is preferred that the primersare from 12 to 60 nucleotides long.

B. Conformation Dependent Labels

Conformation dependent labels refer to all labels that produce a changein fluorescence intensity or wavelength based on a change in the form orconformation of the molecule or compound with which the label isassociated. Examples of conformation dependent labels used in thecontext of probes and primers include molecular beacons, Amplifluors,FRET probes, cleavable FRET probes, TaqMan probes, scorpion primers,fluorescent triplex oligos including but not limited to triplexmolecular beacons or triplex FRET probes, fluorescent water-solubleconjugated polymers, PNA probes and QPNA probes. Such labels, and, inparticular, the principles of their function, can be adapted for usewith the disclosed methods. Several types of conformation dependentlabels are reviewed in Schweitzer and Kingsmore, Curr. Opin. Biotech.12:21-27 (2001).

Stem quenched labels, a form of conformation dependent labels, arefluorescent labels positioned on a nucleic acid such that when a stemstructure forms a quenching moiety is brought into proximity such thatfluorescence from the label is quenched. When the stem is disrupted, thequenching moiety is no longer in proximity to the fluorescent label andfluorescence increases. Examples of this effect can be found inmolecular beacons, fluorescent triplex oligos, triplex molecularbeacons, triplex FRET probes, and QPNA probes, the operationalprinciples of which can be adapted for use with the disclosed methods.

Stem activated labels, a form of conformation dependent labels, arelabels or pairs of labels where fluorescence is increased or altered byformation of a stem structure. Stem activated labels can include anacceptor fluorescent label and a donor moiety such that, when theacceptor and donor are in proximity (when the nucleic acid strandscontaining the labels form a stem structure), fluorescence resonanceenergy transfer from the donor to the acceptor causes the acceptor tofluoresce. Stem activated labels are typically pairs of labelspositioned on nucleic acid molecules such that the acceptor and donorare brought into proximity when a stem structure is formed in thenucleic acid molecule. If the donor moiety of a stem activated label isitself a fluorescent label, it can release energy as fluorescence(typically at a different wavelength than the fluorescence of theacceptor) when not in proximity to an acceptor (that is, when a stemstructure is not formed). When the stem structure forms, the overalleffect would then be a reduction of donor fluorescence and an increasein acceptor fluorescence. FRET probes are an example of the use of stemactivated labels, the operational principles of which can be adapted foruse with the disclosed methods.

C. Detection Labels

To aid in detection and quantitation of microRNAs, detection labels canbe incorporated into detection probes or detection molecules or directlyincorporated into amplied nucleic acids. As used herein, a detectionlabel is any molecule that can be associated with nucleic acid, directlyor indirectly, and which results in a measurable, detectable signal,either directly or indirectly. Many such labels are known to those ofskill in the art. Examples of detection labels suitable for use in thedisclosed method are radioactive isotopes, fluorescent molecules,phosphorescent molecules, enzymes, antibodies, and ligands.

Examples of suitable fluorescent labels include fluoresceinisothiocyanate (FITC), 5,6-carboxymethyl fluorescein, Texas red,nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride,rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin, BODIPY®,Cascade Blue®, Oregon Green®, pyrene, lissamine, xanthenes, acridines,oxazines, phycoerythrin, macrocyclic chelates of lanthanide ions such asquantum Dye™, fluorescent energy transfer dyes, such as thiazoleorange-ethidium heterodimer, and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5and Cy7. Examples of other specific fluorescent labels include3-Hydroxypyrene 5,8,10-Tri Sulfonic acid, 5-Hydroxy Tryptamine (5-HT),Acid Fuchsin, Alizarin Complexon, Alizarin Red, Allophycocyanin,Aminocoumarin, Anthroyl Stearate, Astrazon Brilliant Red 4G, AstrazonOrange R, Astrazon Red 6B, Astrazon Yellow 7 GLL, Atabrine, Auramine,Aurophosphine, Aurophosphine G, BAO 9 (Bisaminophenyloxadiazole), BCECF,Berberine Sulphate, Bisbenzamide, Blancophor FFG Solution, BlancophorSV, Bodipy F1, Brilliant Sulphoflavin FF, Calcien Blue, Calcium Green,Calcofluor RW Solution, Calcofluor White, Calcophor White ABT Solution,Calcophor White Standard Solution, Carbostyryl, Cascade Yellow,Catecholamine, Chinacrine, Coriphosphine O, Coumarin-Phalloidin, CY3.18, CY5.1 8, CY7, Dans (1-Dimethyl Amino Naphaline 5 Sulphonic Acid),Dansa (Diamino Naphtyl Sulphonic Acid), Dansyl NH—CH3, Diamino PhenylOxydiazole (DAO), Dimethylamino-5-Sulphonic acid, DipyrrometheneboronDifluoride, Diphenyl Brilliant Flavine 7GFF, Dopamine, Erythrosin ITC,Euchrysin, FIF (Formaldehyde Induced Fluorescence), Flazo Orange, Fluo3, Fluorescamine, Fura-2, Genacryl Brilliant Red B, Genacryl BrilliantYellow 10GF, Genacryl Pink 3G, Genacryl Yellow 5GF, Gloxalic Acid,Granular Blue, Haematoporphyrin, Indo-1, Intrawhite Cf Liquid, LeucophorPAF, Leucophor SF, Leucophor WS, Lissamine Rhodamine B200 (RD200),Lucifer Yellow CH, Lucifer Yellow VS, Magdala Red, Marina Blue, MaxilonBrilliant Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF, MPS (MethylGreen Pyronine Stilbene), Mithramycin, NBD Amine, Nitrobenzoxadidole,Noradrenaline, Nuclear Fast Red, Nuclear Yellow, Nylosan BrilliantFlavin E8G, Oxadiazole, Pacific Blue, Pararosaniline (Feulgen), PhorwiteAR Solution, Phorwite BKL, Phorwite Rev, Phorwite RPA, Phosphine 3R,Phthalocyanine, Phycoerythrin R, Polyazaindacene Pontochrome Blue Black,Porphyrin, Primuline, Procion Yellow, Pyronine, Pyronine B, PyrozalBrilliant Flavin 7GF, Quinacrine Mustard, Rhodamine 123, Rhodamine 5GLD, Rhodamine 6G, Rhodamine B, Rhodamine B 200, Rhodamine B Extra,Rhodamine BB, Rhodamine BG, Rhodamine WT, Serotonin, Sevron BrilliantRed 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B, Sevron Orange,Sevron Yellow L, SITS (Primuline), SITS (Stilbene Isothiosulphonicacid), Stilbene, Snarf 1, sulpho Rhodamine B Can C, Sulpho Rhodamine GExtra, Tetracycline, Thiazine Red R, Thioflavin S, Thioflavin TCN,Thioflavin 5, Thiolyte, Thiozol Orange, Tinopol CBS, True Blue,Ultralite, Uranine B, Uvitex SFC, Xylene Orange, and XRITC.

Useful fluorescent labels are fluorescein(5-carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine(5,6-tetramethyl rhodamine), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5and Cy7. The absorption and emission maxima, respectively, for thesefluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm;588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm;778 nm), thus allowing their simultaneous detection. Other examples offluorescein dyes include 6-carboxyfluorescein (6-FAM),2′,4′,1,4,-tetrachlorofluorescein (TET),2′,4′,5′,7′,1,4-hexachlorofluorescein (HEX),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyrhodamine (JOE),2′-chloro-5′-fluoro-7′,8′-fused phenyl-1,4-dichloro-6-carboxyfluorescein(NED), and 2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC).Fluorescent labels can be obtained from a variety of commercial sources,including Amersham Pharmacia Biotech, Piscataway, N.J.; MolecularProbes, Eugene, Oreg.; and Research Organics, Cleveland, Ohio.

Additional labels of interest include those that provide for signal onlywhen the probe with which they are associated is specifically bound to atarget molecule, where such labels include: “molecular beacons” asdescribed in Tyagi & Kramer, Nature Biotechnology (1996) 14:303 and EP 0070 685 B1. Other labels of interest include those described in U.S.Pat. No. 5,563,037; WO 97/17471 and WO 97/17076.

Labeled nucleotides are a useful form of detection label for directincorporation into expressed nucleic acids during synthesis. Examples ofdetection labels that can be incorporated into nucleic acids includenucleotide analogs such as BrdUrd (5-bromodeoxyuridine, Hoy and Schimke,Mutation Research 290:217-230 (1993)), aminoallyldeoxyuridine (Henegariuet al., Nature Biotechnology 18:345-348 (2000)), 5-methylcytosine (Sanoet al., Biochim. Biophys. Acta 951:157-165 (1988)), bromouridine(Wansick et al., J. Cell Biology 122:283-293 (1993)) and nucleotidesmodified with biotin (Langer et al., Proc. Natl. Acad. Sci. USA 78:6633(1981)) or with suitable haptens such as digoxygenin (Kerkhof, Anal.Biochem. 205:359-364 (1992)). Suitable fluorescence-labeled nucleotidesare Fluorescein-isothiocyanate-dUTP, Cyanine-3-dUTP and Cyanine-5-dUTP(Yu et al., Nucleic Acids Res., 22:3226-3232 (1994)). A preferrednucleotide analog detection label for DNA is BrdUrd (bromodeoxyuridine,BrdUrd, BrdU, BUdR, Sigma-Aldrich Co). Other useful nucleotide analogsfor incorporation of detection label into DNA are AA-dUTP(aminoallyl-deoxyuridine triphosphate, Sigma-Aldrich Co.), and5-methyl-dCTP (Roche Molecular Biochemicals). A useful nucleotide analogfor incorporation of detection label into RNA is biotin-16-UTP(biotin-16-uridine-5′-triphosphate, Roche Molecular Biochemicals).Fluorescein, Cy3, and Cy5 can be linked to dUTP for direct labeling.Cy3.5 and Cy7 are available as avidin or anti-digoxygenin conjugates forsecondary detection of biotin- or digoxygenin-labeled probes.

Detection labels that are incorporated into nucleic acid, such asbiotin, can be subsequently detected using sensitive methods well-knownin the art. For example, biotin can be detected usingstreptavidin-alkaline phosphatase conjugate (Tropix, Inc.), which isbound to the biotin and subsequently detected by chemiluminescence ofsuitable substrates (for example, chemiluminescent substrate CSPD:disodium,3-(4-methoxyspiro-[1,2,-dioxetane-3-2′-(5′-chloro)tricyclo[3.3.1.1^(3,7)]decane]-4-yl)phenyl phosphate; Tropix, Inc.). Labels can also be enzymes, such asalkaline phosphatase, soybean peroxidase, horseradish peroxidase andpolymerases, that can be detected, for example, with chemical signalamplification or by using a substrate to the enzyme which produces light(for example, a chemiluminescent 1,2-dioxetane substrate) or fluorescentsignal.

Molecules that combine two or more of these detection labels are alsoconsidered detection labels. Any of the known detection labels can beused with the disclosed probes, tags, molecules and methods to label anddetect microRNAs or nucleic acid produced in the disclosed methods.Methods for detecting and measuring signals generated by detectionlabels are also known to those of skill in the art. For example,radioactive isotopes can be detected by scintillation counting or directvisualization; fluorescent molecules can be detected with fluorescentspectrophotometers; phosphorescent molecules can be detected with aspectrophotometer or directly visualized with a camera; enzymes can bedetected by detection or visualization of the product of a reactioncatalyzed by the enzyme; antibodies can be detected by detecting asecondary detection label coupled to the antibody. As used herein,detection molecules are molecules which interact with a compound orcomposition to be detected and to which one or more detection labels arecoupled.

D. Sequence Similarities

It is understood that as discussed herein the use of the terms homologyand identity mean the same thing as similarity. Thus, for example, ifthe use of the word homology is used between two sequences (non-naturalsequences, for example) it is understood that this is not necessarilyindicating an evolutionary relationship between these two sequences, butrather is looking at the similarity or relatedness between their nucleicacid sequences. Many of the methods for determining homology between twoevolutionarily related molecules are routinely applied to any two ormore nucleic acids or proteins for the purpose of measuring sequencesimilarity regardless of whether they are evolutionarily related or not.

In general, it is understood that one way to define any known variantsand derivatives or those that might arise, of the disclosed microRNAsherein, is through defining the variants and derivatives in terms ofhomology to specific known sequences. This identity of particularsequences disclosed herein is also discussed elsewhere herein. Ingeneral, variants of microRNAs herein disclosed typically have at least,about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percenthomology to a stated sequence or a native sequence. Those of skill inthe art readily understand how to determine the homology of two proteinsor nucleic acids, such as genes. For example, the homology can becalculated after aligning the two sequences so that the homology is atits highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison can beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment. It isunderstood that any of the methods typically can be used and that incertain instances the results of these various methods can differ, butthe skilled artisan understands if identity is found with at least oneof these methods, the sequences would be said to have the statedidentity.

For example, as used herein, a sequence recited as having a particularpercent homology to another sequence refers to sequences that have therecited homology as calculated by any one or more of the calculationmethods described above. For example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingthe Zuker calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by any of theother calculation methods. As another example, a first sequence has 80percent homology, as defined herein, to a second sequence if the firstsequence is calculated to have 80 percent homology to the secondsequence using both the Zuker calculation method and the Pearson andLipman calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by the Smith andWaterman calculation method, the Needleman and Wunsch calculationmethod, the Jaeger calculation methods, or any of the other calculationmethods. As yet another example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingeach of calculation methods (although, in practice, the differentcalculation methods will often result in different calculated homologypercentages).

E. Hybridization and Selective Hybridization

The term hybridization typically means a sequence driven interactionbetween at least two nucleic acid molecules, such as a primer or a probeand a microRNA. Sequence driven interaction means an interaction thatoccurs between two nucleotides or nucleotide analogs or nucleotidederivatives in a nucleotide specific manner. For example, G interactingwith C or A interacting with T are sequence driven interactions.Typically sequence driven interactions occur on the Watson-Crick face orHoogsteen face of the nucleotide. The hybridization of two nucleic acidsis affected by a number of conditions and parameters known to those ofskill in the art. For example, the salt concentrations, pH, andtemperature of the reaction all affect whether two nucleic acidmolecules will hybridize.

Parameters for selective hybridization between two nucleic acidmolecules are well known to those of skill in the art. For example, insome embodiments selective hybridization conditions can be defined asstringent hybridization conditions. For example, stringency ofhybridization is controlled by both temperature and salt concentrationof either or both of the hybridization and washing steps. For example,the conditions of hybridization to achieve selective hybridization caninvolve hybridization in high ionic strength solution (6×SSC or 6×SSPE)at a temperature that is about 12-25° C. below the Tm (the meltingtemperature at which half of the molecules dissociate from theirhybridization partners) followed by washing at a combination oftemperature and salt concentration chosen so that the washingtemperature is about 5° C. to 20° C. below the Tm. The temperature andsalt conditions are readily determined empirically in preliminaryexperiments in which samples of reference DNA immobilized on filters arehybridized to a labeled nucleic acid of interest and then washed underconditions of different stringencies. Hybridization temperatures aretypically higher for DNA-RNA and RNA-RNA hybridizations. The conditionscan be used as described above to achieve stringency, or as is known inthe art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989;Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which is hereinincorporated by reference for material at least related to hybridizationof nucleic acids). A preferable stringent hybridization condition for aDNA:DNA hybridization can be at about 68° C. (in aqueous solution) in6×SSC or 6×SSPE followed by washing at 68° C. Stringency ofhybridization and washing, if desired, can be reduced accordingly as thedegree of complementarity desired is decreased, and further, dependingupon the G-C or A-T richness of any area wherein variability is searchedfor. Likewise, stringency of hybridization and washing, if desired, canbe increased accordingly as homology desired is increased, and further,depending upon the G-C or A-T richness of any area wherein high homologyis desired, all as known in the art.

Another way to define selective hybridization is by looking at theamount (percentage) of one of the nucleic acids bound to the othernucleic acid. For example, in some embodiments selective hybridizationconditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid isbound to the non-limiting nucleic acid. Typically, the non-limitingnucleic acid is in for example, 10 or 100 or 1000 fold excess. This typeof assay can be performed at under conditions where both the limitingand non-limiting nucleic acids are for example, 10 fold or 100 fold or1000 fold below their k_(d), or where only one of the nucleic acidmolecules is 10 fold or 100 fold or 1000 fold or where one or bothnucleic acid molecules are above their k_(d).

Another way to define selective hybridization is by looking at thepercentage of nucleic acid that gets enzymatically manipulated underconditions where hybridization is required to promote the desiredenzymatic manipulation. For example, in some embodiments selectivehybridization conditions would be when at least about, 60, 65, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the nucleic acidis enzymatically manipulated under conditions which promote theenzymatic manipulation, for example if the enzymatic manipulation is DNAextension, then selective hybridization conditions would be when atleast about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100percent of the nucleic acid molecules are extended. Preferred conditionsalso include those suggested by the manufacturer or indicated in the artas being appropriate for the enzyme performing the manipulation.

Just as with homology, it is understood that there are a variety ofmethods herein disclosed for determining the level of hybridizationbetween two nucleic acid molecules. It is understood that these methodsand conditions can provide different percentages of hybridizationbetween two nucleic acid molecules, but unless otherwise indicatedmeeting the parameters of any of the methods would be sufficient. Forexample if 80% hybridization was required and as long as hybridizationoccurs within the required parameters in any one of these methods it isconsidered disclosed herein.

It is understood that those of skill in the art understand that if acomposition or method meets any one of these criteria for determininghybridization either collectively or singly it is a composition ormethod that is disclosed herein.

F. Nucleic Acids

There are a variety of molecules disclosed herein that are nucleic acidbased, including, for example, microRNAs. The disclosed nucleic acidscan be made up of for example, nucleotides, nucleotide analogs, ornucleotide substitutes. Non-limiting examples of these and othermolecules are discussed herein. It is understood that for example, whena vector is expressed in a cell, that the expressed mRNA will typicallybe made up of A, C, G, and U. Likewise, it is understood that if anucleic acid molecule is introduced into a cell or cell environmentthrough for example exogenous delivery, it is advantageous that thenucleic acid molecule be made up of nucleotide analogs that reduce thedegradation of the nucleic acid molecule in the cellular environment.

So long as their relevant function is maintained, primers, probes, andany other oligonucleotides and nucleic acids can be made up of orinclude modified nucleotides (nucleotide analogs). Many modifiednucleotides are known and can be used in oligonucleotides and nucleicacids. A nucleotide analog is a nucleotide which contains some type ofmodification to either the base, sugar, or phosphate moieties.Modifications to the base moiety would include natural and syntheticmodifications of A, C, G, and T/U as well as different purine orpyrimidine bases, such as uracil-5-yl, hypoxanthin-9-yl (I), and2-aminoadenin-9-yl. A modified base includes but is not limited to5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Additional basemodifications can be found for example in U.S. Pat. No. 3,687,808,Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and Sanghvi, Y. S., Chapter 15, Antisense Research andApplications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRCPress, 1993. Certain nucleotide analogs, such as 5-substitutedpyrimidines, 6-azapyrimidines and N2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine can increase the stability of duplex formation. Othermodified bases are those that function as universal bases. Universalbases include 3-nitropyrrole and 5-nitroindole. Universal basessubstitute for the normal bases but have no bias in base pairing. Thatis, universal bases can base pair with any other base. Basemodifications often can be combined with for example a sugarmodification, such as 2′-O-methoxyethyl, to achieve unique propertiessuch as increased duplex stability. There are numerous U.S. patents suchas U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941,which detail and describe a range of base modifications. Each of thesepatents is herein incorporated by reference in its entirety, andspecifically for their description of base modifications, theirsynthesis, their use, and their incorporation into oligonucleotides andnucleic acids.

Nucleotide analogs can also include modifications of the sugar moiety.Modifications to the sugar moiety would include natural modifications ofthe ribose and deoxyribose as well as synthetic modifications. Sugarmodifications include but are not limited to the following modificationsat the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-,S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl andalkynyl can be substituted or unsubstituted C1 to C10, alkyl or C2 toC10 alkenyl and alkynyl. 2′ sugar modifications also include but are notlimited to —O[(CH₂)n O]m CH₃, —O(CH₂)n OCH₃, —O(CH₂)n NH₂, —O(CH₂)n CH₃,—O(CH₂)n —ONH₂, and —O(CH₂)n ON[(CH₂)n CH₃)]₂, where n and m are from 1to about 10.

Other modifications at the 2′ position include but are not limited to:C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. Similar modifications canalso be made at other positions on the sugar, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide. Modifiedsugars would also include those that contain modifications at thebridging ring oxygen, such as CH₂ and S, Nucleotide sugar analogs canalso have sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. There are numerous U.S. patents that teach thepreparation of such modified sugar structures such as U.S. Pat. Nos.4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873;5,670,633; and 5,700,920, each of which is herein incorporated byreference in its entirety, and specifically for their description ofmodified sugar structures, their synthesis, their use, and theirincorporation into nucleotides, oligonucleotides and nucleic acids.

Nucleotide analogs can also be modified at the phosphate moiety.Modified phosphate moieties include but are not limited to those thatcan be modified so that the linkage between two nucleotides contains aphosphorothioate, chiral phosphorothioate, phosphorodithioate,phosphotriester, aminoalkylphosphotriester, methyl and other alkylphosphonates including 3′-alkylene phosphonate and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates. It is understood that these phosphate or modifiedphosphate linkages between two nucleotides can be through a 3′-5′linkage or a 2′-5′ linkage, and the linkage can contain invertedpolarity such as 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixedsalts and free acid forms are also included. Numerous U.S. patents teachhow to make and use nucleotides containing modified phosphates andinclude but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of whichis herein incorporated by reference its entirety, and specifically fortheir description of modified phosphates, their synthesis, their use,and their incorporation into nucleotides, oligonucleotides and nucleicacids.

It is understood that nucleotide analogs need only contain a singlemodification, but can also contain multiple modifications within one ofthe moieties or between different moieties.

Nucleotide substitutes are molecules having similar functionalproperties to nucleotides, but which do not contain a phosphate moiety,such as peptide nucleic acid (PNA). Nucleotide substitutes are moleculesthat will recognize and hybridize to (base pair to) complementarynucleic acids in a Watson-Crick or Hoogsteen manner, but which arelinked together through a moiety other than a phosphate moiety.Nucleotide substitutes are able to conform to a double helix typestructure when interacting with the appropriate target nucleic acid.

Nucleotide substitutes are nucleotides or nucleotide analogs that havehad the phosphate moiety and/or sugar moieties replaced. Nucleotidesubstitutes do not contain a standard phosphorus atom. Substitutes forthe phosphate can be for example, short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatom and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts. Numerous U.S. patents disclose how to makeand use these types of phosphate replacements and include but are notlimited to U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134;5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257;5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086;5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704;5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of whichis herein incorporated by reference its entirety, and specifically fortheir description of phosphate replacements, their synthesis, their use,and their incorporation into nucleotides, oligonucleotides and nucleicacids.

It is also understood in a nucleotide substitute that both the sugar andthe phosphate moieties of the nucleotide can be replaced, by for examplean amide type linkage (aminoethylglycine) (PNA). U.S. Pat. Nos.5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNAmolecules, each of which is herein incorporated by reference. (See alsoNielsen et al., Science 254:1497-1500 (1991)).

Oligonucleotides and nucleic acids can be comprised of nucleotides andcan be made up of different types of nucleotides or the same type ofnucleotides. For example, one or more of the nucleotides in anoligonucleotide can be ribonucleotides, 2′-O-methyl ribonucleotides, ora mixture of ribonucleotides and 2′-O-methyl ribonucleotides; about 10%to about 50% of the nucleotides can be ribonucleotides, 2′-O-methylribonucleotides, or a mixture of ribonucleotides and 2′-O-methylribonucleotides; about 50% or more of the nucleotides can beribonucleotides, 2′-O-methyl ribonucleotides, or a mixture ofribonucleotides and 2′-O-methyl ribonucleotides; or all of thenucleotides are ribonucleotides, 2′-O-methyl ribonucleotides, or amixture of ribonucleotides and 2′-β-methyl ribonucleotides. Sucholigonucleotides and nucleic acids can be referred to as chimericoligonucleotides and chimeric nucleic acids.

G. Solid Supports

Solid supports are solid-state substrates or supports with whichmolecules (such as probes) or other components used in, or produced by,the disclosed methods can be associated. Molecules can be associatedwith solid supports directly or indirectly. For example, probes can bebound to the surface of a solid support. An array is a solid support towhich multiple probes or other molecules have been associated in anarray, grid, or other organized pattern.

Solid-state substrates for use in solid supports can include any solidmaterial with which components can be associated, directly orindirectly. This includes materials such as acrylamide, agarose,cellulose, nitrocellulose, glass, gold, polystyrene, polyethylene vinylacetate, polypropylene, polymethacrylate, polyethylene, polyethyleneoxide, polysilicates, polycarbonates, teflon, fluorocarbons, nylon,silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid,polyorthoesters, functionalized silane, polypropylfumerate, collagen,glycosaminoglycans, and polyamino acids. Solid-state substrates can haveany useful form including thin film, membrane, bottles, dishes, fibers,woven fibers, shaped polymers, particles, beads, microparticles, or acombination. Solid-state substrates and solid supports can be porous ornon-porous. A chip is a rectangular or square small piece of material.Preferred forms for solid-state substrates are thin films, beads, orchips. A useful form for a solid-state substrate is a microtiter dish.In some embodiments, a multiwell glass slide can be employed.

An array can include a plurality of molecules, compounds or probesimmobilized at identified or predefined locations on the solid support.Each predefined location on the solid support generally has one type ofcomponent (that is, all the components at that location are the same).Alternatively, multiple types of components can be immobilized in thesame predefined location on a solid support. Each location will havemultiple copies of the given components. The spatial separation ofdifferent components on the solid support allows separate detection andidentification.

Although useful, it is not required that the solid support be a singleunit or structure. A set of molecules, compounds and/or probes can bedistributed over any number of solid supports. For example, at oneextreme, each component can be immobilized in a separate reaction tubeor container, or on separate beads or microparticles.

Methods for immobilization of oligonucleotides to solid-state substratesare well established. Oligonucleotides, including address probes anddetection probes, can be coupled to substrates using establishedcoupling methods. For example, suitable attachment methods are describedby Pease et al., Proc. Natl. Acad. Sci. USA 91(11):5022-5026 (1994), andKhrapko et al., Mol Biol (Mosk) (USSR) 25:718-730 (1991). A method forimmobilization of 3′-amine oligonucleotides on casein-coated slides isdescribed by Stimpson et al., Proc. Natl. Acad. Sci. USA 92:6379-6383(1995). A useful method of attaching oligonucleotides to solid-statesubstrates is described by Guo et al., Nucleic Acids Res. 22:5456-5465(1994).

Each of the components immobilized on the solid support can be locatedin a different predefined region of the solid support. The differentlocations can be different reaction chambers. Each of the differentpredefined regions can be physically separated from each other of thedifferent regions. The distance between the different predefined regionsof the solid support can be either fixed or variable. For example, in anarray, each of the components can be arranged at fixed distances fromeach other, while components associated with beads will not be in afixed spatial relationship. In particular, the use of multiple solidsupport units (for example, multiple beads) will result in variabledistances.

Components can be associated or immobilized on a solid support at anydensity. Components can be immobilized to the solid support at a densityexceeding 400 different components per cubic centimeter. Arrays ofcomponents can have any number of components. For example, an array canhave at least 1,000 different components immobilized on the solidsupport, at least 10,000 different components immobilized on the solidsupport, at least 100,000 different components immobilized on the solidsupport, or at least 1,000,000 different components immobilized on thesolid support.

H. Kits

The materials described above as well as other materials can be packagedtogether in any suitable combination as a kit useful for performing, oraiding in the performance of, the disclosed method. It is useful if thekit components in a given kit are designed and adapted for use togetherin the disclosed method. For example disclosed are kits for detectingand measuring microRNAs, the kit comprising amplification primers anddetection probes. The kits also can contain enzymes and reactionsolutions.

I. Mixtures

Disclosed are mixtures formed by performing or preparing to perform thedisclosed method. For example, disclosed are mixtures comprising a bodyfluid and amplification primers, microRNA and amplification primers, andamplified microRNA and detection probes.

Whenever the method involves mixing or bringing into contactcompositions or components or reagents, performing the method creates anumber of different mixtures. For example, if the method includes 3mixing steps, after each one of these steps a unique mixture is formedif the steps are performed separately. In addition, a mixture is formedat the completion of all of the steps regardless of how the steps wereperformed. The present disclosure contemplates these mixtures, obtainedby the performance of the disclosed methods as well as mixturescontaining any disclosed reagent, composition, or component, forexample, disclosed herein.

J. Systems

Disclosed are systems useful for performing, or aiding in theperformance of, the disclosed method. Systems generally comprisecombinations of articles of manufacture such as structures, machines,devices, and the like, and compositions, compounds, materials, and thelike. Such combinations that are disclosed or that are apparent from thedisclosure are contemplated. For example, disclosed and contemplated aresystems comprising microRNAs and a detection apparatus.

K. Data Structures and Computer Control

Disclosed are data structures used in, generated by, or generated from,the disclosed method. Data structures generally are any form of data,information, and/or objects collected, organized, stored, and/orembodied in a composition or medium. A temporal pattern of microRNAlevels stored in electronic form, such as in RAM or on a storage disk,is a type of data structure.

The disclosed method, or any part thereof or preparation therefor, canbe controlled, managed, or otherwise assisted by computer control. Suchcomputer control can be accomplished by a computer controlled process ormethod, can use and/or generate data structures, and can use a computerprogram. Such computer control, computer controlled processes, datastructures, and computer programs are contemplated and should beunderstood to be disclosed herein.

L. Actions Based on Identifications

The disclosed methods include the determination, identification,indication, correlation, diagnosis, prognosis, etc. (which can bereferred to collectively as “identifications”) of subjects, diseases,conditions, states, etc. based on measurements, detections, comparisons,analyses, assays, screenings, etc. For example, levels or amounts ofmicroRNAs can be used to identify subjects that have or are at risk ofmyocardial infarction, left ventricular remodeling, left ventricularhypertrophy, diastolic heart failure, aortic aneurysm, ischemia, and/orelectrical stimulation. Such identifications are useful for manyreasons. For example, and in particular, such identifications allowspecific actions to be taken based on, and relevant to, the particularidentification made. For example, diagnosis of a particular disease orcondition in particular subjects (and the lack of diagnosis of thatdisease or condition in other subjects) has the very useful effect ofidentifying subjects that would benefit from treatment, actions,behaviors, etc. based on the diagnosis. For example, treatment for aparticular disease or condition in subjects identified is significantlydifferent from treatment of all subjects without making such anidentification (or without regard to the identification). Subjectsneeding or that could benefit from the treatment will receive it andsubjects that do not need or would not benefit from the treatment willnot receive it.

Accordingly, also disclosed herein are methods comprising takingparticular actions following and based on the disclosed identifications.For example, disclosed are methods comprising creating a record of anidentification (in physical—such as paper, electronic, or other—form,for example). Thus, for example, creating a record of an identificationbased on the disclosed methods differs physically and tangibly frommerely performing a measurement, detection, comparison, analysis, assay,screen, etc. Such a record is particularly substantial and significantin that it allows the identification to be fixed in a tangible form thatcan be, for example, communicated to others (such as those who couldtreat, monitor, follow-up, advise, etc. the subject based on theidentification); retained for later use or review; used as data toassess sets of subjects, treatment efficacy, accuracy of identificationsbased on different measurements, detections, comparisons, analyses,assays, screenings, etc., and the like. For example, such uses ofrecords of identifications can be made, for example, by the sameindividual or entity as, by a different individual or entity than, or acombination of the same individual or entity as and a differentindividual or entity than, the individual or entity that made the recordof the identification. The disclosed methods of creating a record can becombined with any one or more other methods disclosed herein, and inparticular, with any one or more steps of the disclosed methods ofidentification.

As another example, disclosed are methods comprising making one or morefurther identifications based on one or more other identifications. Forexample, particular treatments, monitorings, follow-ups, advice, etc.can be identified based on the other identification. For example,identification of subject as having a disease or condition with a highlevel of a particular component can be further identified as a subjectthat could or should be treated with a therapy based on or directed tothe high level component. A record of such further identifications canbe created (as described above, for example) and can be used in anysuitable way. Such further identifications can be based, for example,directly on the other identifications, a record of such otheridentifications, or a combination. Such further identifications can bemade, for example, by the same individual or entity as, by a differentindividual or entity than, or a combination of the same individual orentity as and a different individual or entity than, the individual orentity that made the other identifications. The disclosed methods ofmaking a further identification can be combined with any one or moreother methods disclosed herein, and in particular, with any one or moresteps of the disclosed methods of identification.

As another example, disclosed are methods comprising treating,monitoring, following-up with, advising, etc. a subject identified inany of the disclosed methods. Also disclosed are methods comprisingtreating, monitoring, following-up with, advising, etc. a subject forwhich a record of an identification from any of the disclosed methodshas been made. For example, particular treatments, monitorings,follow-ups, advice, etc. can be used based on an identification and/orbased on a record of an identification. For example, a subjectidentified as having a disease or condition with a high level of aparticular component (and/or a subject for which a record has been madeof such an identification) can be treated with a therapy based on ordirected to the high level component. Such treatments, monitorings,follow-ups, advice, etc. can be based, for example, directly onidentifications, a record of such identifications, or a combination.Such treatments, monitorings, follow-ups, advice, etc. can be performed,for example, by the same individual or entity as, by a differentindividual or entity than, or a combination of the same individual orentity as and a different individual or entity than, the individual orentity that made the identifications and/or record of theidentifications. The disclosed methods of treating, monitoring,following-up with, advising, etc. can be combined with any one or moreother methods disclosed herein, and in particular, with any one or moresteps of the disclosed methods of identification.

Uses

The disclosed methods and compositions are applicable to numerous areasincluding, but not limited to, diagnose, assess prognosis, monitorimprovement or deterioration, or monitor the progress of treatment ofmyocardial infarction, cardiac failure, cardiac hypertrophy, leftventricular remodeling, or a combination. Other uses include determiningif and when a myocardial infarction occurred. Other uses are disclosed,apparent from the disclosure, and/or will be understood by those in theart.

Methods

Disclosed are methods and materials for assessing cardiac failure,cardiac hypertrophy, and left ventricular remodeling using microRNAlevels. The level of microRNAs can be measured in a body fluid, such asplasma and serum. Disclosed is method comprising detecting one or moretarget microRNAs in a body fluid of a subject at a plurality ofdifferent times. The temporal pattern of the level of the one or moretarget microRNAs can indicates the presence, severity, or a combinationof left ventricular remodeling in the subject.

The presence, severity, or a combination of left ventricular remodelingin the subject can be indicated by comparing the temporal pattern of thelevel of the one or more target microRNAs to one or more referencetemporal patterns. The one or more microRNAs can comprise one or more ofmiR-1, miR-21, miR-23a, miR-29a, miR-30, miR-133a, miR-150, miR-195,miR-199, miR-208, miR-214, and miR-125b. The one or more microRNAs cancomprise one or more of miR-1, miR-21, miR-29a, miR-133a, miR-208, andmiR-125b. The one or more microRNAs can comprise one or more of miR-1,miR-21, miR-29a, miR-133a, and miR-208.

The body fluid can be, for example, blood, plasma, serum, or lymphaticfluid. The plurality of different times at which the one or moremicroRNAs are detected can comprise two or more times separated by 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64,65, 70, 75, 80, 81, 82, 83, 84 85, 86, 87, 88, 89, and 90 days. Theplurality of different times at which the one or more microRNAs aredetected can comprise two or more times separated by 1, 2, 3, 4, 5, 10,15, 20, 23, 24, 25, 26, 27, 28, 30, 35, 40, 45, 50, 55, 60, 62, 65, 70,75, 80, 85, 86, 87, 88, 89, and 90 days. The plurality of differenttimes at which the one or more microRNAs are detected can comprise twoor more times separated by 2, 3, 23, and 62 days. The plurality ofdifferent times at which the one or more microRNAs are detected cancomprise two or more times that are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, 65, 70, 75, 80, 81, 82, 83,84 85, 86, 87, 88, 89, and 90 days following a known or suspectedmyocardial infarction. The plurality of different times at which the oneor more microRNAs are detected can comprise two or more times that are1, 2, 3, 4, 5, 10, 15, 20, 23, 24, 25, 26, 27, 28, 30, 35, 40, 45, 50,55, 60, 62, 65, 70, 75, 80, 85, 86, 87, 88, 89, and 90 days following aknown or suspected myocardial infarction. The plurality of differenttimes at which the one or more microRNAs are detected can comprise twoor more times that are 2, 3, 23, and 62 days following a known orsuspected myocardial infarction. The level of the one or more targetmicroRNAs can comprise the measured level of the one or more targetmicroRNAs normalized to the measured level of a reference RNA in thebody fluid. The reference RNA can be snRNA U6.

The level of the one or more target microRNAs can comprise the measuredlevel of the one or more target microRNAs expressed as the folddifference of the measured level of the one or more target microRNAs tothe measured level of the one or more target microRNAs in a referencesubject. The level of the one or more target microRNAs can comprise themeasured level of the one or more target microRNAs normalized to themeasured level of a reference RNA in the body fluid expressed as thefold difference of the normalized level of the one or more targetmicroRNAs to the measured level of the one or more target microRNAs inthe same body fluid of reference subject normalized to the measuredlevel of a reference RNA in the body fluid of the reference subject.

The level of the one or more target microRNAs in a reference subject canbe measured at the same time as the level of the one or more targetmicroRNAs is measured in the subject. The level of the one or moretarget microRNAs in a reference subject can be measured at a differenttime than the level of the one or more target microRNAs is measured inthe subject. The level of the one or more target microRNAs in areference subject can be a reference level.

The plurality of different times can comprise two or more times 1, 2, 3,4, 5, 10, 15, 20, 25, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, and 90 days following a known or suspected myocardial infarction inthe subject. The plurality of different times can comprise two or moretimes 2, 5, 28, and 90 days following a known or suspected myocardialinfarction in the subject. The temporal pattern of the level of the oneor more target microRNAs can indicate that the subject suffered amyocardial infarction. The temporal pattern of the level of the one ormore target microRNAs can indicate how long ago the subject suffered themyocardial infarction.

Examples of temporal patterns of microRNAs following myocardialinfarction are shown in FIGS. 4-8, 26, and 27. For example, a level oramount in a subject of miR-1, miR-208, miR-133a, miR-21, miR-29a, or acombination, higher than the level or amount in control, reference,and/or normal subjects and/or in a control or reference RNA in thesubject or in control, reference, and/or normal subjects indicatesmyocardial infarction, left ventricular remodeling, and/or leftventricular hypertrophy. As another example, a level or amount in asubject of miR-29a, higher than the level or amount in control,reference, and/or normal subjects and/or in a control or reference RNAin the subject or in control, reference, and/or normal subjectsimmediately after a suspected myocardial infarction indicates myocardialinfarction, left ventricular remodeling, and/or left ventricularhypertrophy. The reference RNA can be, for example, snRNA U6.

Disclosed are methods comprising detecting one or more target microRNAsin tissue or a body fluid of a subject, where the level or amount of theone or more target microRNAs indicates, for example, the risk,development, presence, severity, or a combination, of thoracic aorticaneurysm in the subject, of diastolic heart failure in the subject, ofleft ventricular hypertrophy in the absence of diastolic heart failurein the subject, of left ventricular remodeling in the subject, that thesubject experienced ischemia-reperfusion, or a combination.

MicroRNAs can be used to assess or diagnose aortic aneurysm. Forexample, a level or amount in a subject of miR-133a, miR-21, miR-29a,miR-208, or a combination, lower than the level or amount in control,reference, and/or normal subjects and/or in a control or reference RNAin the subject or in control, reference, and/or normal subjectsindicates aortic aneurysm. The control or reference level can be, forexample, the level in normal aorta.

In some forms the methods can indicate the risk, presence, severity, ora combination, of thoracic aortic aneurysm in the subject. In someforms, the one or more target microRNAs can comprise miR-1, miR-21,miR-29a, miR-133a, and miR-486-5p, or a combination, wherein a level oramount of miR-1, miR-21, miR-29a, miR-133a, and miR-486-5p, or acombination, less than the level or amount in control subjects,reference subjects, normal subjects, or a combination, indicates therisk, presence, severity, or a combination, of thoracic aortic aneurysmin the subject.

In some forms, a set of microRNAs is detected and if a thresholdpercentage or more of the set of microRNAs has an indicated level oramount, the subject can be assessed for a disease or condition. Forexample, if a threshold percentage or more of a set of microRNAs has alevel or amount of less than or greater than (depending on theindividual microRNA) than the level or amount in control subjects,reference subjects, normal subjects, or a combination, it indicates arisk, presence, severity, or a combination, of thoracic aortic aneurysmin a subject.

In some forms, the one or more target microRNAs comprises miR-455-3p,miR-1268, miR-338-5p, miR-940, miR-1323, miR-768-3p, miR-574-3p,miR-106b, miR-451, miR-100, miR-125b, miR-195, miR-19b, miR-30d,miR-15b, miR-125a-5p, miR-143, miR-193b, miR-16, miR-27a, miR-29a,miR-30a, miR-27b, miR-92a, miR-140-5p, let-7i, miR-151-5p, miR-140-3p,miR-24, miR-23a, miR-145, miR-199b-3p, miR-199a-3p, miR-361-5p,miR-130a, miR-22, and miR-497. For miR-455-3p, miR-1268, miR-338-5p, andmiR-940, a level or amount greater than the level or amount in controlsubjects, reference subjects, normal subjects, or a combination, iscorrelated with the risk, presence, severity, or a combination, ofthoracic aortic aneurysm in the subject. For miR-1323, miR-768-3p,miR-574-3p, miR-106b, miR-451, miR-100, miR-125b, miR-195, miR-19b,miR-30d, miR-15b, miR-125a-5p, miR-143, miR-193b, miR-16, miR-27a,miR-29a, miR-30a, miR-27b, miR-92a, miR-140-5p, let-7i, miR-151-5p,miR-140-3p, miR-24, miR-23a, miR-145, miR-199b-3p, miR-199a-3p,miR-361-5p, miR-130a, miR-22, and miR-497, a level or amount less thanthe level or amount in control subjects, reference subjects, normalsubjects, or a combination, is correlated with the risk, presence,severity, or a combination, of thoracic aortic aneurysm in the subject.

In some forms, the threshold percentage can be on or about 60%, 65%,70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more of the target microRNAs.

In some forms, the one or more target microRNAs can comprise miR-1,miR-21, miR-29a, and miR-133a, or a combination, wherein the lower thelevel or amount of miR-1, miR-21, miR-29a, miR-133a, and miR-486-5p, ora combination, less than the level or amount in control subjects,reference subjects, normal subjects, or a combination, the larger thethoracic aortic aneurysm in the subject.

In some forms, the lower the level or amount of miR-29a less than thelevel or amount in control subjects, reference subjects, normalsubjects, or a combination, the higher the level of MMP-2 in thesubject. In some forms, the lower the level or amount of miR-133a lessthan the level or amount in control subjects, reference subjects, normalsubjects, or a combination, the higher the level of MMP-9 in thesubject. In some forms, prior to detection, the subject can be suspectedof having thoracic aortic aneurysm.

In some forms, decreased level (compared to normal) of miR-1, miR-21,miR-29a, miR-133a, and miR-486-5p is correlated with TAA (decreaseindicates TAA). In some forms, the magnitude of decreased level(compared to normal) of miR-1, miR-21, miR-29a, and miR-133a iscorrelated with size of TAA (larger decrease indicates larger TAA). Insome forms, the level (compared to normal) of miR-29a is inverselycorrelated with MMP-2 (decreased level of miR-29a indicates increasedlevel of MMP-2). In some forms, the level (compared to normal) ofmiR-133a is inversely correlated with MMP-9 (decreased level of miR-133aindicates increased level of MMP-9). In some forms, a decreased level of33 specific miRNAs and/or an increased level of 4 specific miRNAs arecorrelated with TAA (decrease indicates TAA).

In some forms the methods can indicate the risk, development, presence,severity, or a combination, of diastolic heart failure in a subjecthaving left ventricular hypertrophy. In some forms, the one or moretarget microRNAs can comprise miR-1, miR-21, miR-29a, miR-133a, andmiR-760, or a combination, wherein a decrease in the level or amount ofmiR-1, miR-21, miR-29a, miR-133a, and miR-760, or a combination, from alevel or amount greater than the level or amount in control subjects,reference subjects, normal subjects, or a combination, indicates therisk, development, presence, severity, or a combination, of diastolicheart failure in the subject.

In some forms, the one or more target microRNAs comprises miR-1, miR-21,miR-29a, miR-133a, and miR-760, or a combination, wherein a decrease inthe level or amount of miR-1, miR-21, miR-29a, miR-133a, and miR-760, ora combination, from a level or amount in subjects with left ventricularhypertrophy but not diastolic heart failure, indicates the risk,development, presence, severity, or a combination, of diastolic heartfailure in the subject.

The decrease in the level or amount of the one or more target microRNAscan be determined by detecting the level or amount of the one or moretarget microRNAs at different times. The level or amount of the one ormore target microRNAs at one or more of the different times can begreater than the level or amount in control subjects, referencesubjects, normal subjects, or a combination.

In some forms the methods can indicate the presence or absence ofdiastolic heart failure in a subject having left ventricularhypertrophy. In some forms, the one or more target microRNAs cancomprise miR-1, miR-21, miR-29a, miR-133a, and miR-760, or acombination, wherein a level or amount of miR-1, miR-21, miR-29a,miR-133a, and miR-760, or a combination, about the same as the level oramount in control subjects, reference subjects, normal subjects, or acombination, indicates the presence of diastolic heart failure in thesubject.

In some forms, the one or more target microRNAs comprises miR-1, miR-21,miR-29a, miR-133a, and miR-760, or a combination, wherein a level oramount of miR-1, miR-21, miR-29a, miR-133a, and miR-760, or acombination, greater than the level or amount in control subjects,reference subjects, normal subjects, or a combination, indicates theabsence of diastolic heart failure in the subject.

In some forms, increased level (compared to normal) of miR-1, miR-21,miR-29a, miR-133a, and miR-760 is correlated with LVH without DHF(increase indicates LVH without DHF). In some forms, unchanged level(compared to normal) of miR-1, miR-21, miR-29a, miR-133a, and miR-760 iscorrelated with LVH with DHF (normal in LVH patients indicates LVH withDHF). In some forms, change in level from increased (compared to normal)of miR-1, miR-21, miR-29a, miR-133a, and miR-760 is correlated withdevelopment of DHF in LVH patients (return to normal in LVH patientsindicates LVH with DHF).

In some forms the methods can indicate that the subject experiencedischemia-reperfusion. In some forms, the one or more target microRNAscan comprise miR-1, miR-133a, miR-760, or a combination, wherein a levelor amount of miR-1, miR-133a, miR-760, or a combination, greater thanthe level or amount in control subjects, reference subjects, normalsubjects, or a combination, indicates that the subject experiencedischemia-reperfusion. In some forms, the subject can be suspected ofhaving experienced ischemia-reperfusion. In some forms, increased level(compared to normal) of miR-1, miR-133a, and miR-760 is correlated withischemia-reperfusion (increase indicates I/R).

As another example, a level or amount in a subject of miR-1, miR-208,miR-133a, miR-21, miR-29a, or a combination, higher than the level oramount in control, reference, and/or normal subjects and/or in a controlor reference RNA in the subject or in control, reference, and/or normalsubjects followed by a level or amount in the subject of miR-1, miR-21,miR-29a, miR-133a, miR-760, or a combination, lower than the level oramount of miR-1, miR-21, miR-29a, miR-133a, miR-760, respectively,indicates development of diastolic heart failure. As another example, ina subject with left ventricular remodeling but not diastolic heartfailure, a level or amount in the subject of miR-1, miR-21, miR-29a,miR-133a, miR-760, or a combination, lower than the level or amount ofmiR-1, miR-21, miR-29a, miR-133a, miR-760, respectively, measuredearlier in the subject indicates development of diastolic heart failure.

In some forms the methods can indicate a higher risk of left ventricularremodeling in the subject. In some forms, the one or more targetmicroRNAs can be detected following myocardial infarction.

In some forms, the one or more target microRNAs can comprise miR-1,miR-21, or a combination, wherein the one or more target microRNAs aredetected on or about 2 days following myocardial infarction, wherein alevel or amount of miR-1, miR-21, or a combination, less than the levelor amount in control subjects, reference subjects, normal subjects, or acombination, indicates a higher risk of left ventricular remodeling inthe subject.

In some forms the methods can indicate a higher risk of increased leftventricular end diastolic volume developing in the subject. In someforms, the one or more target microRNAs can comprise miR-29a, whereinthe one or more target microRNAs are detected on, about, or between 4and 10 days following myocardial infarction, wherein a level or amountof miR-29a more than the level or amount in control subjects, referencesubjects, normal subjects, or a combination, indicates a higher risk ofincreased left ventricular end diastolic volume developing in thesubject.

In some forms, the one or more target microRNAs can be detected on,about, or between 5 and 8 days following myocardial infarction. In someforms, the one or more target microRNAs can be detected on or about 5days following myocardial infarction.

In some forms, miR-1 and miR-21 levels decreased (compared to normal) at2 days post-MI correlated with LVR. In some forms, miR-29a levels(compared to normal) early after MI (e.g., day 5) is correlated withleft ventricular end diastolic volume (LV EDV) late after MI (e.g., day90).

In some forms, the one or more target microRNAs are detected in atissue. The tissue can be cardiac tissue. The tissue can be thoracicaortic tissue. In some forms, the one or more target microRNAs aredetected in a body fluid. The body fluid can be plasma. In some forms,the one or more target microRNAs can be detected in a tissue sample or abody fluid sample collected from the subject. In some forms, detectionof the one or more microRNAs can be accomplished by altering the tissueor body fluid so that the microRNAs produce a signal. In some forms, thealteration of the tissue or body sample comprising amplifying themicroRNAs. For example, the microRNAs can be amplified via polymerasechain reaction (PCR). In some forms, the alteration can be hybridizationof a probe to the microRNAs or amplified copies of the microRNAsequences.

In some forms, the level of the one or more target microRNAs cancomprise the measured level of the one or more target microRNAsnormalized to the measured level of a reference RNA in the tissue orbody fluid. In some forms, the reference RNA can be snRNA U6.

In some forms, the level of the one or more target microRNAs cancomprise the measured level of the one or more target microRNAsexpressed as the fold difference of the measured level of the one ormore target microRNAs to the measured level of the one or more targetmicroRNAs in a reference subject. In some forms, the level of the one ormore target microRNAs can comprise the measured level of the one or moretarget microRNAs normalized to the measured level of a reference RNA inthe tissue or body fluid expressed as the fold difference of thenormalized level of the one or more target microRNAs to the measuredlevel of the one or more target microRNAs in the same tissue or bodyfluid of reference subject normalized to the measured level of areference RNA in the tissue or body fluid of the reference subject.

In some forms, the level of the one or more target microRNAs in areference subject can be measured at the same time as the level of theone or more target microRNAs is measured in the subject. In some forms,the level of the one or more target microRNAs in a reference subject canbe measured at a different time than the level of the one or more targetmicroRNAs is measured in the subject. In some forms, the level of theone or more target microRNAs in a reference subject can be a referencelevel.

In some forms, the method can further comprise treating the subject forthoracic aortic aneurysm. In some forms, the method can further comprisetreating the subject for diastolic heart failure. In some forms, themethod can further comprise treating the subject forischemia-reperfusion injury. In some forms, the method can furthercomprise treating the subject for left ventricular remodeling.

“Risk” or like terms refers to the chance, probability, likelihood, etc.that an event, characteristic, condition, etc. will be present or willoccur. Risk can be expressed, for example, numerically, quantitatively,qualitatively, as a ratio, as a percentage, or in other appropriateways. Risk can be expressed with or without reference to other subjects,states, averages, means, medians, and/or quartile values of thereference values.

“Indirect measurement” or like terms refer to a measurement that isrepresentative of something else. For example, the number of PCR cyclesbefore inflection of amplification can indirectly indicate the amount ofthe original template being amplified.

“Reference level” or like terms refers to a level established from asample(s) from a subject(s) that is considered a control. A referencelevel could be, for example, from healthy individuals or from subjectsnot suffering cardiac disease or condition (or the relevant cardiacdisease or condition). It is understood that the reference level can beproduced de novo or can be a number previously determined as a referencelevel. Generally, by “cells . . . of similar type to the cell” is meantthat the first cells are the same type of cells as the second cells,similar to the type of the second cells, where “type of cells” can referto the cell type of the cells, the tissue type of the cells, the organfrom which the cells come, or a combination.

“Greater” or like terms refers to more of something than in a comparisonvalue, composition, component, etc. Various measures can be greater thanother measures. “Higher” or like terms refers to more of something thanin a comparison value, composition, component, etc. Various measures canbe higher than other measures. “larger” or like terms refers tosomething of greater dimension, extent, and/or volume than in acomparison object, composition, component, etc. Various objects,dimensions, and/or volumes can be larger than other etc. Variousobjects, dimensions, and/or volumes.

“Less” or like terms refers to less or fewer of something than in acomparison value, composition, component, etc. Various measures can beless than other measures. “Lower” or like terms refers to less or fewerof something than in a comparison value, composition, component, etc.Various measures can be lower than other measures. “Decrease” or liketerms refers to less or fewer of something than in a comparison value,composition, component, etc. Various measures can decrease compared toother measures.

“Subject” or like terms refers to an individual. Thus, the “subject” caninclude, for example, domesticated animals, such as cats, dogs, etc.,livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratoryanimals (e.g., mouse, rabbit, rat, guinea pig, etc.) mammals, non-humanmammals, primates, non-human primates, rodents, birds, reptiles,amphibians, fish, and any other animal. The subject can be a mammal suchas a primate or a human. The subject can also be a non-human.

“Sample” or like terms refers to an animal, a plant, a fungus, etc.; anatural product, a natural product extract, etc.; a tissue or organ froman animal; a cell (either within a subject, taken directly from asubject, or a cell maintained in culture or from a cultured cell line);a cell lysate (or lysate fraction) or cell extract; or a solutioncontaining one or more molecules derived from a cell or cellularmaterial (e.g. a polypeptide or nucleic acid), which is assayed asdescribed herein. A sample may also be any body fluid or excretion (forexample, but not limited to, blood, serum, plasma, lymphatic fluid,mucus, urine, stool, saliva, tears, bile) that contains cells or cellcomponents. For the disclosed method, the sample can be from an animal,such as a mammal, such as a human.

“Treatment” or “treating” or like terms refer to the medical managementof a subject with the intent to cure, ameliorate, stabilize, or preventa disease, pathological condition, or disorder. This term includesactive treatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder. It is understood that treatment, while intendedto cure, ameliorate, stabilize, or prevent a disease, pathologicalcondition, or disorder, need not actually result in the cure,ameliorization, stabilization or prevention. The effects of treatmentcan be measured or assessed as described herein and as known in the artas is suitable for the disease, pathological condition, or disorderinvolved. Such measurements and assessments can be made in qualitativeand/or quantitative terms. Thus, for example, characteristics orfeatures of a disease, pathological condition, or disorder and/orsymptoms of a disease, pathological condition, or disorder can bereduced to any effect or to any amount.

“Patient history” or “subject history” or like terms refers to one ormore items in the history of a subject which could be consideredrelevant to the subject, such as race, age, gender, physical status,such as pre- or post-menopausal, pregnant, diabetic, overweight orobese, smoking, alcohol consumption, family cancer history, or likeitems.

“Fresh whole blood” refers to a sample of venous blood from a subjectand was kept at 4° C. for less than 48 hours.

“Fluorescent” or like terms refers to luminescence that is caused by theabsorption of radiation at one wavelength followed by nearly immediatereradiation usually at a different wavelength and that ceases almost atonce when the incident radiation stops, as understood in the art.

“Obtaining” or like terms refers to getting or acquiring. For example,obtaining a sample includes taking a sample physically from a subjectand it also includes receiving a sample which someone else took from asubject, which was for example, stored. Thus, obtaining includes but isnot limited to physically collecting a sample.

EXAMPLES A. Example 1 Temporal Patterns of miRNA in Plasma FollowingMyocardial Infarction

Left ventricular (LV) end-diastolic volume (EDV) was measured byechocardiography on day 1, day 28, and day 90 post-MI. Plasma miRNAswere measured in age matched normal (NML, n=6) and post-MI patients(n=12) from day 2 to day 90 post-MI. All MI patients received standardtherapy. Day 1 measurements were preformed within 72 hours of MI. Thepeak troponin was 167±31. ANOVA and prcomp pairwise on log transformeddata.

Plasma RNA (100 uL) was isolated and miRNA reverse transcription forstem loop primers corresponding to specific miRNAs was performed. ThemiRNAs measured were miR-1, miR-21, miR-29a, miR-125b, miR-133a, andmiR-208. The resultant cDNA was subjected to pre-amplification (10cycles) and corresponding miRNA real-time primers. RT-PCR wasperformedand data normalized to endogenous snRNA U6. This approachprovided high sensitivity, linearity, and reproducibility. Followingmyocardial infarction, Left ventricular end-diastolic volume (LV EDV)increased progressively compared to the age matched normals. This wasaccompanied by time dependent changes in specific miRNAs (Table 1). Forexample, miRNA initially fell at 2 days post-MI, then increased 2-foldover the age matched normals 5 days post-MI, and returned to the levelof age matched normals 90 days post-MI. In contrast, miR-133a andmiR-208 increased 5 days post-MI and remained elevated 90 days post-MI.

Determinants of LV remodeling include insufficient or maladaptivehypertrophy, increase apoptosis, and altered extracellular matrixstructure. MicroRNAs are small noncoding RNAS (approximately 22nucleotides long) that control gene expression. The miRNAS target mRNAfor degradation and/or translational repression. MicroRNAs are importantregulators of normal growth and disease dependent altered growthregulation. The disclosed methods represent a sensitive, reliable methodto measure miRNAs in plasma, measure serial or temporal changes inspecific miRNAs following a myocardial infarction, and assess thetemporal relationship between changes in miRNAs and LV remodeling inpatients following a myocardial infarction.

MicroRNA was isolated from plasma by isolating plasma RNA (100 uL) usingmirVana Paris kit (Ambion). miRNA was reverse transcribed to cDNA. ThecDNA was subjected to a 10-cycle pre-amplification step (AppliedBiosystems). Quantitative PCR was then performed using TaqMan primerscorresponding to the target (miR-1, miR-21, miR-29a, miR-208, miR-133a)and control (snRNA U6) RNAs. The results are shown in FIGS. 12 and14-19. The temporal pattern of plasma miRNA is shown in FIGS. 4-8. Foldchange was shown relative to miRNA levels from age matched normalsubjects. Levels of miRNA were normalized using measured levels of snRNAU6 (used as a standard control). Quantitative PCR results for snRNA U6are shown in FIG. 11. Statistical analysis of the results is shown inFIG. 13.

A unique temporal pattern of miRNAs occurred in post-MI patientsincluding changes in miRNAs previously shown to regulate myocardialgrowth, fibrosis, and remodeling. Thus, serially profiling miRNAs in theplasma of post-MI patients can be used to diagnose, assess, and monitorremodeling in patients.

The results show that a unique temporal patterns of miRNAs previouslyshown to regulate myocaridal growth, fibrosis, and remodeling arepresent and reliably detectable in post-myocardial infarction patients.The temporal patterns are correlated to the level and severity of leftventricular remodeling after myocardial infarction (FIG. 9) indicatingthat the disclosed methods can be used for detecting, diagnosing,monitoring, prognosing cardiac remodeling and assessing theeffectiveness of treatments for myocardial infarction and remodeling.

B. Example 2 Relationship Between The Temporal Profile of PlasmamicroRNA and Left Ventricular Remodeling In Patients FollowingMyocardial Infarction

1. Summary

MicroRNAs (miRs) are small noncoding RNAs that associate with targetmRNAs, act as negative regulators of gene expression by promoting mRNAdegradation or inhibiting translation, and play a regulatory role inmyocardial growth, fibrosis, viability, and remodeling. Whether specifictemporal changes in miRs occur in patients during the LV remodelingprocess that follows a myocardial infarction (post-MI) remains unknown.The current study shows that plasma miRs can be reliably measured inpost-MI patients and that there is a relationship between temporalchanges in specific miRs and post-MI LV structural remodeling.

LV end-diastolic volume (EDV) and plasma miRs (miR-1, -21, -29a, 133a,208, quantitative rt-PCR, normalized for endogenous snRNA U6) weremeasured in referent controls (CTL n=6) and post-MI patients (n=12) fromday 2 through day 90 post-MI. Following MI, EDV increased progressivelycompared to CTL; this was accompanied by time dependent changes inspecific miRs. For example, miR-21 (inhibits apoptosis) initially fell 2days post-MI increased 5 days post-MI and returned to CTL values atlater time points. In contrast, miR-29a (inhibits changes in theextracellular matrix) increased 5 days post-MI and then fell to CTL.miR-208 (augments hypertrophy) increased 5 days post-MI and remainedelevated.

In conclusion, a unique temporal pattern of miRs occurred in post-MIpatients that included an early and robust rise in miRs that have beenshown to affect myocardial growth, fibrosis and viability. Thus,serially profiling miRs in the plasma of post-MI patients can hold bothmechanistic and prognostic significance.

2. Introduction

Left ventricular remodeling represents the aggregate effects of changesin cardiomyocytes, fibroblasts, and interstitial structure and functionthat result from cardiovascular disease processes such as a myocardialinfarction. The molecular regulatory mechanisms that affect cellular andextracellular remodeling remain incompletely defined; however, recentstudies suggest that microRNAs (miRs) may be one such mechanism(Divakaran V, Mann DL. The Emerging Role of MicroRNAs in CardiacRemodeling and Heart Failure. Circ Res. 2008; 103:1072-1083; Small E M,Frost R J A, Olson E N. MicroRNAs Add a New Dimension to CardiovascularDisease. Circulation. 2010; 121:1022-1032). MiRs are small noncodingRNAs (˜22 nucleotides) that associate with target mRNAs and act as anegative regulator of gene expression by promoting mRNA degradation orinhibiting translation. Studies in animal models have suggested thatmiRs play a translational or post-translational regulatory role inmyocardial growth, fibrosis, viability, and remodeling (Liu et al.microRNA-133a regulates cardiomyocyte proliferation and suppressessmooth muscle gene expression in the heart. Genes Dev. 2008;22(23):3242-54; Duisters et al. miR-133 and miR-30 Regulate ConnectiveTissue Growth Factor. Implications for a Role of MicroRNAs in MyocardialMatrix Remodeling. Circ Res. 2009; 104:170-178; van Rooij et al.Dysregulation of microRNAs after myocardial infarction reveals a role ofmiR-29 in cardiac fibrosis. Proc Natl Acad. Sci. 2008; 105(35):13027-32;Dong et al. MicroRNA Expression Signature and the Role of MicroRNA-21 inthe Early Phase of Acute Myocardial Infarction. J Biol. Chem. 2009;284(43):29514-25; Roy et al. MicroRNA expression in response to murinemyocardial infarction: miR-21 regulates fibroblast metalloprotease-2 viaphosphatase and tensin homologue. Cardiovasc Res. 2009; 82(1):21-9). Forexample, miR-1 have been suggested to blunt LV hypertrophy, augmentapoptosis and facilitate progressive dilation, miR-208 augmentshypertrophy and increases the extracellular matrix, miR-21 and miR-133ainhibit apoptosis and miR-29a inhibits changes in the extracellularmatrix.

Whether specific temporal changes in miRs occur in patients during theLV remodeling process that follows a myocardial infarction (post-MI)remains unknown. It is impractical to assess serial changes in miRs inpost-MI patients using repetitive LV myocardial tissue biopsies;however, this can be done using plasma sampling. Accordingly, thecurrent study disclosed herein developed a sensitive, reliable method tomeasure miRs in plasma in referent control subjects and post-MIpatients, and to measure serial changes in specific miRs following an MIto determine whether there is a relationship between temporal changes inspecific miRs and LV structural remodeling in post-MI patients.

3. Methods

i. Protocol

Twelve patients with a confirmed MI (Post-MI) and 6 referent age-matchedcontrol subjects (CTL) were enrolled in this study after providinginformed consent. All of the studies described herein were reviewed andapproved by the Medical University of South Carolina InstitutionalReview Board.

For the MI patients, studies were performed beginning at the time ofenrollment (post-MI day 1). Plasma from a peripheral vein blood samplewas used to measure miR profiles at post-MI days 2, 5, 28, and 90. Atpost-MI days 1, 5, 28, and 90 an echocardiogram was obtained. For thereferent control subjects, an echocardiogram and plasma sample wasperformed once at the time of enrollment. All subjects fasted overnightbefore each study but took their morning medications as prescribed.

Transthoracic echocardiography was performed using a Sonos 5500 systemwith an S-4 MHz transducer (Agilent Technologies, Andover, Mass.).Measurements were made with American Society of Echocardiographycriteria.

ii. Subjects

Twelve patients with a confirmed MI and 6 referent age-matched controlsubjects were enrolled in this study. An ECG and/or a positive cardiacenzyme panel confirmed the MI. Patients were excluded from enrollment ifthere was a history of a previous MI; previous coronaryrevascularization surgery within past 24 months; a history of activemalignancy; significant renal or hepatic dysfunction; activerheumatological disease. MI patient were treated according to AHA/ACCguidelines. The referent control group consisted of subjects with noevidence of cardiovascular disease. Cardiovascular disease was excludedby performing a complete medical history, comprehensive physicalexamination, ECG, and echocardiogram.

By experimental design there were no differences in age between referentcontrol and post-MI patients (Table 3). The ratio of men to women washigher in the post-MI group. Heart rate and blood pressure werecomparable between groups. Differences in medications reflect expectedACC/AHA guideline based protocols for post-MI patients. In the referentcontrol subjects, β-adrenergic blockers, angiotensin-converting enzymeinhibitors, and angiotensin receptor antagonists were used to treat mildincreases in systolic pressure. Aspirin or anti-inflammatory agents wereused for management for arthritic pain.

TABLE 3 Demographics for Referent Control Subjects and MyocardialInfarction Patients Control MI Number 6 12 Age (years) 59 ± 2 58 ± 3Males 2 (33%) 9(75%) * Body Surface Area (m2)  1.87 ± 0.03  1.99 ± 0.04Heart Rate (bpm) 70 ± 1 68 ± 2 Arterial Systolic Pressure (mmHg) 126 ±4  119 ± 4  Arterial Diastolic Pressure (mmHg) 75 ± 3 67 ± 3

iii. Plasma miRNA Measurements

Small RNAs from plasma were isolated using the mirVana PARIS Kit(AM1556, Ambion) which is based upon a denaturing/phenol chloroformextraction approach. Briefly, 400 μL of plasma was added to an equalamount of denaturing solution, and incubated on ice for 5 minutes.Following which, 800 μL of an acid-phenol chloroform solution was addedto the samples in order to inactivate RNAases and to create an aqueousRNA phase. This aqueous phase was removed, and passed throughglass-fiber filters binding the RNA. The RNA was then eluted using a lowionic-strength solution, yielding a final volume of 100 μL. Then, 11.4μL was reversed transcribed into cDNA (Applied Biosystems TaqManMicroRNA RT Kit #4366579) using pre-specified miR sequences for: miR-1,miR-29a, miR-133a, miR-21, miR-208, and snRNA U6 (Table 1). Next, 12.5μL of the cDNA was preamplified (TaqMan PreAmp Master Mix Kit #4391128,Applied Biosystems) as well as the pooled miR primers. Finally, thepreamplification product was subjected to real time PCR (CFX96 Real-TimeSystem, BioRad). The relative cycle threshold (Ct) values for U6 snRNAwere used as endogenous controls for normalizing the respective miR Ctvalues (Li et al. Real-Time Polymerase Chain Reaction MicroRNA DetectionBased on Enzymatic Stem-Loop Probes Ligation. Anal Chem. 2009 Jul. 1;81(13):5446-51) and were calculated as dCt (dCt=miRNA Ct-snRNA U6 Ct).Changes in miRNA were reported as a fold change from referent controlcalculated as FC=2̂^((dCt post MI-dCt referent control)). Thesenormalized Ct values were computed for each sample, and thesemeasurements were performed in duplicate. Referent control values wereset at 1.0. Therefore, in post-MI patients fold change values less then1.0 represented a fall in miRNA expression and fold change valuesgreater then 1.0 represented an increase in miRNA expression compared toreferent control. In initial assays performed in triplicate usingreferent control samples, the coefficient of variation for individualmiR values was less than 10% (Table 2).

TABLE 1  Applied Biosystems miRNA primers SEQ Catalog miRNA ID NO:Number Target Sequence miR-1 1 2222 UGGAAUGUAAAGAAGUAUGUAU miR-21 2 0397UAGCUUAUCAGACUGAUGUUGA miR-29a 3 2112 UAGCACCAUCUGAAAUCGGUUA miR-133a 42246 UUUGGUCCCCUUCAACCAGCUG miR-208a 5 0511 AUAAGACGAGCAAAAAGCUUGUU6 snRNA 6 1973 GUGCUCGCUUCGGCAGCACAUA UACUAAAAUUGGAACGAUACAGAGAAGAUUAGCAUGGCCCCUGCG CAAGGAUGACACGCAAAUUCGUG AAGCGUUCCAUAUUUUUACUGCCCUCCAUGCCCUGCCCCACAAACG CUCUGAUAACAGUCUGUCCCUGU CUCUCUCCUGCUGCUCCUAUGGAAGCGAAGUUUUCCGCUCCUGCAG AAAGCAAAGUUACGACUCAGAGAC GGCUGAGGAUGACAUCAGCGAUGUGC

TABLE 2 Ct Values and Coefficient of Variation for Referent ControlsmiRNA Ct Values (Mean ± SEM) Coefficient of Variation (%) miR-1 30.62 ±0.38 2.80% miR-21 22.77 ± 0.20 8.18% miR-29a 25.44 ± 0.52 9.29% miR-133a30.62 ± 0.31 4.54% miR-208a 36.54 ± 1.65 5.08% U6 snRNA 30.00 ± 0.453.8%

Five miRs and one endogenous control were chosen for this study. Arepresentative miR was chosen to target a translational orpost-translational molecular regulatory role in each aspect of post-MIremodeling including augmenting or inhibiting hypertrophy, extracellularmatrix changes, apoptosis, and progressive dilation. miR-1 has beensuggested to blunt LV hypertrophy, augment apoptosis and facilitate toprogressive dilation, miR-208 augments hypertrophy and increases changesin the extracellular matrix, miR-21 and miR-133a inhibit apoptosis andmiR-29a inhibits changes in the extracellular matrix.

iv. Data Analysis

The echocardiographic and miRNA data were presented in an untransformedmanner using parametric statistics. Comparisons between CTL values andpost-MI values were examined using a 2-way ANOVA for repeated measuresin which CTL/Post-MI was the first treatment level and time after MI wasthe second treatment level. After the ANOVA, pair-wise comparisons weremade using a Bonferroni method. The relationship between changes in miRlevels and LV volumes in the post-MI period were examined by linearregression methods. Values of p<0.05 were considered significant. Allvalues are presented as the mean and SEM. Statistical procedures wereperformed with Stata Statistical Software (StataCorp, release 8.0,College Station, Tex.). The authors had full access to the data and takefull responsibility for their integrity. All authors have read and agreeto the manuscript as written.

4. Results

i. LV Structure

LV end-diastolic volume increased in a time-dependent manner in thepost-MI group as shown in FIG. 24. LV end diastolic volume was alreadyincreased compared with referent control on day 1 post MI. LVend-diastolic volumes increased further from post-MI day 1 values atpost-MI day 28 and 90.

ii. miR

Examples of Qrt-PCR results for miR-1 and snRNA U6 in a referent controlsubject and an MI patient 5 days post MI are shown in FIG. 25. miR-1expression was increased 5 days post MI as evidenced by a significantdecrease in Ct from 31 in the referent control to 28 in the 5 day postMI patient. By comparison no change in snRNA U6 was seen post MIcompared to referent control.

There were time dependent changes in the 5 measured miRs in the post MIpatients compared to the referent control subjects (FIGS. 26-27). miR-1and miR-21 fell at day 2 post MI, miR-29a, miR-133a and miR-208 wereunchanged at day 2 post MI. miR-1, miR-133a, and miR-208 increased atday 5 and remained elevated through day 90 post MI. miR-21 and miR-29awere increased at day 5 but returned to normal by day 90 post MI.

There was a significant association between miR-29a early after MI (postMI day 5) and LVEDV late after an MI (post Mi day 90), r=0.77 and p<0.05(FIG. 27). The greater the increase in miR-29a at 5 days post MI, thegreater the increase in LVEDV at 90 days post-MI.

5. Discussion

The principle finding in this study are three fold. First, miRs can bereproducibly measured in the plasma of patients following a myocardialinfarction using a sensitive, reliable method. Second, differential miRexpression occurred following a myocardial infarction, particularly inthose miRs that are associated with myocardial growth, fibrosis andviability. Third, a unique temporal pattern of miRNAs occurred inpost-MI patients. Therefore, serially profiling miRs in the plasma ofpost-MI patients can have both mechanistic and prognostic significance.

i. miR Processing and Function

miRs are synthesized and processed in the nucleus, then transported intothe cytoplasm and further processed into mature miRs (Divakaran V, MannDL. The Emerging Role of MicroRNAs in Cardiac Remodeling and HeartFailure. Circ Res. 2008; 103:1072-1083; Small et al. MicroRNAs Add a NewDimension to Cardiovascular Disease. Circulation. 2010; 121:1022-1032).miRs associate with target mRNAs and act as negative regulators of geneexpression by promoting mRNA degradation or inhibiting translation.Increased expression levels of miRs can also result in the “paradoxical”up regulation of previously suppressed target genes either directly, bydecreasing the expression of inhibitory proteins and/or transcriptionfactors, or indirectly, by inhibiting the expression levels ofinhibitory miRs. Alternatively, decreased expression levels ofinhibitory miRs can lead directly to increased target gene expression.Therefore, miRs are now believed to play a translational orpost-translational regulatory role in myocardial growth, fibrosis,viability, and remodeling in response to cardiovascular disease.

ii. Plasma miRNAs

Given the fact that blood contains ribonucleases (RNases) it might beexpected that neither serum nor plasma should contain any intact RNA.However, recent studies have demonstrated the presence of miRs in normalsubjects and patients with disease (Mitchell et al. CirculatingmicroRNAs as stable blood-based markers for cancer detection. Proc NatlAcad Sci USA. 2008; 105(30):10513-8; Chen et al. Characterization ofmicroRNAs in serum: a novel class of biomarkers for diagnosis of cancerand other diseases. Cell Research. 2008; 18:997-1006). Further studiesaddressed the question of whether Qrt-PCR products found in plasmasamples were the result of contamination by degraded products of largemolecular weight RNA, tRNA, or genomic DNA. Studies indicate that thereis a stable reproducible population of miR that exists in a form that isresistant to endogenase RNase, possibly because it is packaged inside anexosome or is associated with other molecules. For example, some of thetotal RNA isolated from human plasma was degraded by treatment withexogenous RNase, however, miRs were not degraded. miRs were not degradedby treatment with DNase, multiple freeze thaw cycles, prolongedincubation, or a large range of pH. However, when miRs, not homologousto human miR, were added to human plasma, these miRs were degraded. Inpatients with known cancer in whom tissue samples demonstrate anincrease in specific miRs, the plasma has also been shown to haveincreased miRs.

iii. miRs and LV Remodeling

The role(s) of each miR in these processes remain controversial andincompletely defined; therefore assigning cause and effect relationshipshave not been firmly established. However, current information is basedprimarily on murine models of pressure-overload and MI, heterozygous orhomozygous deletion of miR genes, use of antisense knockdown, and humanmyocardial samples of patients with end stage heart failure. What isclear from these studies is that miRs do contribute to the process of LVremodeling.

Studies have suggested that an increase in miR-1 and -133a maycontribute to adverse remodeling by down regulating calmodulin and MEF2aand attenuating cardiomyocyte hypertrophy and fibrosis. As disclosedherein, increased miR-1 and -133a expression can contribute to theadverse LV remodeling consisting of progressive LV dilation thatcommonly follows an MI. An attenuation of both cardiomyocyte hypertrophyand a high rate of ECM turnover could contribute to this adverseremodeling process.

An increase in miR-21 may increase fibroblast survival, promote MMP-2expression, increase collagen turnover and promote cardiomyocyteapoptosis. Disclosed herein, miR-21 was decreased at 2 days, increasedat 5 days, and returned to normal after 5 days. This pattern reflectsexpected temporal pattern of changes in fibroblast number and activityfollowing an MI, particularly within the infarcted myocardium.

miR-29a targets genes involved in ECM synthesis and turnover includingcollagens, fibrillins and elastin. Disclosed herein, miR-29a wasincreased 5 days post-MI a time during which the most rapid increase inLV volumes occurred, the interstitial matrix would have a high rate ofturnover without significant establishment of mature structuralfibrillar proteins. By contrast, miR-29a returned to normal at timeslater than 5 days post MI as disclosed herein and in previous studies ofend stage ischemic cardiomyopathy, when fibrosis is significant, wasfound to be increased.

iv. Limitations

Quantitation of miR expression patterns using plasma sampling reflectsglobal LV remodeling and can not be used to examine region specificchanges in expression as would be possible for myocardial tissuesamples. On the other hand, plasma samples do provide capability forserial measurements and provide temporal patterns not possible bymyocardial tissue biopsy. In addition, plasma sampling provides anavenue for the use of miRs as diagnostic and prognostic biomarkers. Forexample, data from the study disclosed herein indicates that the extentof increased expression of miR-29a early post-MI is associated with theextent of remodeling late post-MI.

No cause and effect relationships between miR expression and changes incellular and extracellular structure and function can be made based onthe current studies. However, associations between miR temporal patternsand LV structural remodeling were detected.

C. Example 3 Plasma microRNA in Patients with Hypertensive Heart DiseaseDifferential Expression in Left Ventricular Hypertrophy Versus DiastolicHeart Failure

MicroRNAs (miRs) are small noncoding RNAs that associate with targetmRNAs and act as regulators of gene expression by promoting mRNAdegradation or inhibiting translation. Animal models suggest that miRsplay a translational or post-translational regulatory role in myocardialgrowth, fibrosis, and remodeling. This study demonstrates that specificmiRs are differentially expressed in patients with LV hypertrophy (LVH)or diastolic heart failure (DHF). The results show that plasma miRs canbe reliably measured in patients and that there is selective regulationof specific miRs with LVH vs. DHF.

1. Methods and Results

Plasma miR, echocardiography, 6 minute hall walk (6 MHW) were measuredin controls (CTL n=15) and patients with LVH but no DHF (n=13, LVH), andpatients with LVH and DHF (n=13, DHF). DHF patients had shorter 6 MHW,increased pulmonary wedge pressures, and increased nt-proBNP comparedwith CTL or LVH patients. Selected miRs (miR-1, -21, -29a, 133a, 760)were measured using quantitative rt-PCR and normalized for endogenoussnRNA U6 which served as a control (FIG. 20). Coefficient of variationfor all miRs was less than 10%. In LVH, there were increases in miR-21(augment hypertrophic growth), and increased miR-1, 29a, 133a, and 760(limit fibrosis). In DHF, this compensatory response in miRs was lost;all miRs were similar to CTL possibly facilitating the increasedfibrosis and less growth induction characteristic of DHF.

A unique profile of miRs was upregulated in patients with LVH; however,this compensatory response at the level of translational regulation waslost in patients who had developed DHF. See FIG. 20. Changes in miRscould serve as a novel biomarker identifying a molecular signature whichreflects a change in translational regulation in patients making thetransition from hypertrophy to heart failure.

D. Example 4 MicroRNA Profiling in Thoracic Aortic Aneurysm Disease NewDiagnostic and Mechanistic Insights

MicroRNAs (miRs) are short non-coding RNAs that are endogenouslyexpressed and function to inhibit gene expression throughtranscriptional and post-transcription mechanisms. While miR expressionhas been extensively studied in heart disease, their role in regulatinggene expression during thoracic aortic aneurysm (TAA) development hasyet to be explored. Accordingly, the present studied examined theexpression level of seven miRs from patients with ascending TAAs thatfunction to regulate multiple genes involved in aneurysm formation andprogression.

MicroRNA was isolated from aortic tissue specimens, acquired at the timeof surgical resection, from patients with ascending TAAs and tricuspidaortic valves (n=30). The relative expression levels of miR-(1, 21, 29a,133a, 208, 486, and 760) were determined by quantitative real-time PCR.Results (mean±SEM) were expressed as a percent change from a cohort ofnormal aortic specimens (n=10) obtained from patients without aorticdisease. See FIG. 21.

1. Results

Six of the seven miRs were expressed in the ascending thoracic aorta;the myocardial-specific miR-208 was not detected. A significant decrease(p<0.05 versus normal aorta, 100%) in the expression levels of miR-21,29a, and 133a was demonstrated.

The unique findings from this study demonstrate altered miR expressionpatterns in clinical TAA specimens. The decreased miR expression in thisstudy, suggests a loss of inhibitory control of genes regulatingcellular growth/differentiation (miR-21), tissue remodeling (miR-29a,miR-133a), and cellular signaling (miR-133a). Altered miR profilessuggest that these miRs have relevance to the biological and clinicalbehavior of TAAs, and may prove to be useful as biomarkers fordiagnostic or prognostic applications.

E. Example 5 The Human Myocardial Interstitium Contains a SpecificPortfolio of microRNAs which are Dynamically Regulated FollowingIschemia-Reperfusion

MicroRNAs (miRs) regulate post-transcriptional events relevant tomyocardial growth, viability and matrix remodeling (ie miR-1, -21, -29a,-133a. -486, -760). However, direct demonstration that miRs are releasedin a quantifiable manner within human myocardial interstitial fluid(INTf) and dynamically changed following ischemia-reperfusion (I/R)remained defined.

1. Methods and Results

Using novel microdialysis methods and high-sensitivity extraction, INTfwas collected from the mid-myocardium of the LV free wall in patients(n=10, 63±3 yrs, male) undergoing elective coronary revascularizationfor the continuous collection of INTf prior to cardioplegic/myocardialarrest and cardiopulmonary bypass (Baseline) and following cross-clamprelease and reperfusion (POST-I/R), with plasma collected at identicaltime points. See FIG. 22. Absolute miR content was determined byreal-time PCR with a coefficient of variation of less than 10%; where aconsistent yield of the constitutive miR-RNU6B was obtained from bothINTf and plasma samples (31.9±0.4, 29.9±1.2 Ct, respectively). A robustBaseline miR concentration was detected in the INTf as well as plasma(FIG. 22). Higher levels of certain miRs (miR-1, -133a, -760) whichregulate growth and signaling, were in the INTf-indicative of myocardialcompartmentalization. Dynamic changes occurred in both INTf and plasmafollowing I/R (Table), where selective miRs differentially changedwithin the INTf such as miR-1 and -760.

These unique findings demonstrated a robust expression of miRs whichregulate myocyte and matrix remodeling within the human myocardialinterstitium and dynamically change with I/R. In light of the fact thatmiRs form an important control point in transcriptional regulation, thisstudy provides the first clinical evidence that miRs likely form a novelextracellular signaling/regulatory pathway within the intact humanmyocardium.

F. Example 6 High Frequency Electrical Stimulation of Human MyocardialFibroblasts Causes Differential Expression of microRNAs

Basic studies have established that micro RNAs (miRs) can regulatepost-transcriptional processes relevant to myocardial remodeling. Forexample, miRs-21 and 29a have been suggested to regulate fibroblastgrowth and matrix remodeling. However, whether human left ventricularmyocardial fibroblasts (LVMFs) specifically express these miRs and maybe differentially regulated with specific stimuli had remained unclear.This study utilized primary human LVMFs in order to directly quantifymiR-21 and 29a under steady-state conditions and following highfrequency electrical stimulation.

1. Methods/Results

LVMFs from patients with normal function (ejection fraction>=50%, n=5),were grown to 80% confluence. LVMFs were serum deprived for 24 hours,and then randomized to electrical stimulation (6×10⁵ cells/well, 100V 5ms pulses) for 24 hours at 4 Hz (n=6 wells/frequency). See FIG. 23.Unstimulated cells from the same subjects (0 Hz, UNSTIM) served ascontrols. Electrical stimulation had no effect on LVMF viability.Expression levels of the constitutive miRs, snRNA U6B and U44, andmiR-21 and miR-29a were determined by quantitative real-time PCR fromLVMFs. There was high expression of U6B and U44 in LVMF, which wereunaltered by stimulation (CT values U6B: 31±1 vs. 31±1 and U44: 32±1 vs.32±1, respectively). Expression of miR-21 and -29a were normalized forlevels of the constitutive miRs, and expressed as a fold change fromUNSTIM (FIG. 23). With electrical stimulation, relative expression ofmiR-21 was lower while that of miR-29a was more than two-fold higherthan UNSTIM (FIG. 23).

These unique findings of this study are two-fold: First, miR-21 and -29aare highly expressed in human LVMFs and appear to be differentiallyregulated by external stimuli. Second, human LVMFs respond to highfrequency electrical stimulation through differential expression ofmiR-21 and -29a. These findings hold relevance to regulation ofmyocardial extracellular matrix remodeling.

G. Example 7 Selective microRNA Suppression in Human Thoracic AneurysmsRelationship of miR-29a to Aortic Size and Proteolytic Induction

1. Introduction

Within the spectrum of cardiovascular diseases, thoracic aorticaneurysms (TAAs) continue to be one of the most dangerous and difficultto treat problems in cardiothoracic surgery. While it is clear thataortic dysfunction and dilatation are a direct result of pathologicalremodeling of the aortic extracellular matrix (ECM), and that thisprocess is mediated in part by the family of matrix metalloproteinases(MMPs), there remains a paucity of information regarding the upstreammechanisms that regulate these enzymes during TAA development. Recently,a novel class of small non-coding RNA molecules (microRNAs, miRs), 20-25nucleotides in length, were shown to have important post-transcriptionalgene regulatory functions (Bartel D P. MicroRNAs: genomics, biogenesis,mechanism, and function. Cell. 2004; 116(2):281-297). MicroRNAs targetshort nucleotide sequences within the 3′ untranslated region (UTR) ofspecific messenger RNAs (mRNAs), and function to induce messagedegradation, or more typically, translational repression. To date, morethan 1000 unique miRs have been identified within the human genome(miRBase statistics) (Kozomara A, Griffiths-Jones S. miRBase:integrating microRNA annotation and deep-sequencing data. Nucleic AcidsRes. 2011; 39(Database issue):D152-157), and based on computationalmethodology current predictions suggest that approximately one third ofexpressed human genes contain miR regulatory target sites (Lewis B P, etal. Conserved seed pairing, often flanked by adenosines, indicates thatthousands of human genes are microRNA targets. Cell. 2005;120(1):15-20). Moreover, a single miR is capable of targeting multiplemRNAs, and a single mRNA may contain multiple miR binding sites (BartelD P. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136(2):215-233; Friedman R C, et al. Most mammalian mRNAs are conservedtargets of microRNAs. Genome Res. 2009; 19(1):92-105; Krek A, et al.Combinatorial microRNA target predictions. Nat. Genet. 2005;37(5):495-500). Together, this indicates that a common set of miRs canfine-tune the protein abundance of a cassette of specific genes thattogether influence specific cellular functions.

As it is becoming increasingly apparent, miRs are important determinantsof disease within the cardiovascular system. Clear roles for altered miRexpression (Small E M, Frost R J, Olson E N. MicroRNAs add a newdimension to cardiovascular disease. Circulation. 2010;121(8):1022-1032) have been implicated in the regulation of smoothmuscle cell phenotype, angiogenesis, atherosclerosis, restenosis, andother vascular injury responses (Small E M, Frost R J, Olson E N.MicroRNAs add a new dimension to cardiovascular disease. Circulation.2010; 121(8):1022-1032; Albinsson S, et al. MicroRNAs are necessary forvascular smooth muscle growth, differentiation, and function.Arterioscler Thromb Vasc Biol. 2010; 30(6):1118-1126; Qin S, Zhang C.MicroRNAs in vascular disease. J Cardiovasc Pharmacol. 2011; 57(1):8-12;Sen C K, et al. Micromanaging vascular biology: tiny microRNAs play bigband. J Vasc Res. 2009; 46(6):527-540). Moreover, recent work hasidentified that changes in miR expression may contribute to thepathogenesis of aortic aneurysm or dissection. Liu and colleaguesprofiled miR expression in a rat model of abdominal aortic aneurysmusing a miR microarray, and identified 15 dysregulated miRs withputative targets in multiple signaling pathways, including the mitogenactivated protein kinase (MAPK) pathway, which may be important for AAAdevelopment (Liu G, et al. Identification and characteristics ofmicroRNAs with altered expression patterns in a rat model of abdominalaortic aneurysms. Tohoku J Exp Med. 2010; 222(3):187-193). Elia andcoworkers, examined the miR-143/miR-145 cluster in mouse model ofatherosclerosis (ApoE knockout mouse), and reported markedly decreasedexpression levels in the knockout mice, that were further reduced whenthe animals were fed a high fat diet (Elia L, et al. The knockout ofmiR-143 and -145 alters smooth muscle cell maintenance and vascularhomeostasis in mice: correlates with human disease. Cell Death Differ.2009; 16(12):1590-1598). They then examined human biopsies fromascending aortic aneurysms, and again observed a loss of expression formiR-143/miR-145, and suggested that the loss of these miRs inducedstructural modifications in the aorta, mediated in part through theincomplete differentiation of vascular smooth muscle cells. Lastly, Liaoand coworkers recently examined miR expression in aortic tissue from asmall cohort of patients with thoracic aortic dissections using a miRmicroarray (Liao M, et al. A microRNA profile comparison betweenthoracic aortic dissection and normal thoracic aorta indicates thepotential role of microRNAs in contributing to thoracic aorticdissection pathogenesis. J Vasc Surg. 2011;doi:10.1016/j.jvs.2010.11.113). They likewise reported dysregulated miRexpression (18 miRs upregulated, 56 miRs downregulated) with putativetargets in the focal adhesion and MAPK pathways. Together, these reportsindicate that dysregulated miR expression is a common feature ofvascular disease and dysfunction, and that targeting the regulation ofmiR expression/function may provide significant therapeutic advantages.

Accordingly, it is not unreasonable to postulate that alterations in miRexpression can exert an important role in the aortic medial degenerationand dilatation associated with TAA development. Therefore, the presentstudy measured miR expression levels in clinical TAA specimens andexamined the coordinate relationships with indices of aneurysmprogression.

2. Methods

i. Patient Demographics

The study population consisted of aortic tissue specimens obtained fromascending TAA patients with tricuspid aortic valves (n=30) at time ofsurgical resection. The study excluded specimens from patients withMarfan syndrome, bicuspid aortic valves, or documented aorticdissection. Results were compared to a reference control groupconsisting of non-aneurysmal aortic specimens (n=10) collected from theascending aorta of heart donors, and coronary artery bypass graftpatients. There were no gender (Control=80% male, TAA=83% male; p=0.81χ²) or age differences (control=57±4 years, TAA=62±2 years; p=0.31)between control and ascending TAA patients. All aortic specimens weremaintained in frozen storage (−80° C.) until time of experimentation.These specimens are a part of a multi-institutional aortic tissue banklocated at the Medical University of South Carolina. This study wasapproved by the Institutional Review Boards of the Medical University ofSouth Carolina, the University of Pennsylvania, and Yale University, andinformed consent was obtained from all patients.

ii. Sample Preparation and miR Expression Analysis

Resected aortic tissue specimens were homogenized in cell disruptionbuffer (mirVana PARIS miRNA Isolation Kit; Applied Biosystems/Ambion,Austin, Tex.) using a Qiagen Tissuelyser (Qiagen, Valencia, Calif.)bead-mill homogenizer. Total RNA was isolated (mirVana PARIS miRNAIsolation Kit), and reverse transcribed (TaqMan MicroRNA ReverseTranscription Kit; Applied Biosystems). For quantitative PCR studies,the cDNA was pre-amplified according to the manufacturer recommendations(TaqMan PreAmp Master Mix; Applied Biosystems) and the products usedwith specific TaqMan MicroRNA Assays (hsa-miR-1, hsa-miR-21,hsa-miR-29a, hsa-miR-133a-1, hsa-miR-208, hsa-miR-486-5p, hsa-miR-760;and snRNA U6 (internal control); Applied Biosystems) to analyze specificmiR expression on a CFX96 real-time PCR detection system (Bio-Rad,Hercules, Calif.) using the following 2-step procedure: initialdenaturation for 10 min at 95° C., was followed by 40 cycles of 15 secat 95° C., and 60 sec at 60° C.

iii. Microarray analysis

Total RNA isolated from 4 normal aortic specimens and 4 large TAAs wasused for miR expression analysis by microarray. The total RNA,containing low molecular weight (LMW) RNA was examined on a Bioanalyzer2100 (Agilent Technologies) with the Small RNA Chip to ensure LMW RNAcontent and quality. Total RNA samples (100 ng) were labeled with theFlashTag™ Biotin RNA Labeling Kit (Genisphere LLC) followingmanufacturer recommendations. Biotin-labeled samples were hybridized toAffymetrix GeneChip® miRNA Arrays that were then washed, fluorescentlylabeled, and scanned using Affymetrix instrumentation in accordance withAffymetrix and Genisphere protocols. The resulting hybridization datawere processed with Affymetrix miRNA QC Tool software (Version1.0.33.0). Processing settings were as follows: 1) detection scoring wasapplied; 2) background adjustment used the BC-CG Adjust algorithm; 3)normalization was done using the quantile method; 4) the optionalprocesses ‘added small constant’ and ‘threshold’ were used at defaultsettings; 5) summarization was done using median polish. Resultingsummarized hybridization data for the human miR content was thenimported into dChip for comparative analysis (Li C, Wong WH. Model-basedanalysis of oligonucleotide arrays: expression index computation andoutlier detection. Proc Natl Acad Sci USA. 2001; 98(1):31-36).Differential expression was assessed as either a change in detection ora statistical change in magnitude between aneurysmal and controlsamples. Differential detection was assigned to miRs scoring entirelyundetected in one group but detected in one of more samples of the othergroup; differential magnitude was assigned to miRs detected in one ormore samples of both groups and differing by fold change>1.5, p<0.05(Student's t-test, unpaired). False discovery for differential magnitudewas estimated as the median number of human miRs discovered by 100iterations of comparison with randomized sample assignments.

iv. miR Target Prediction

As a first approach to identifying potential miR binding sites in MMP-2and MMP-9, the full-length transcripts for MMP-2 and MMP-9 were used tosearch the TargetScanHuman database (version 5.1, web sitetargetscan.org/). The results identified target binding sites formiR-29a (conserved, 7mer-8; context score=−0.01, P_(CT)=0.82) andmiR-133a (poorly conserved, 7mer-8; context score=−0.04, P_(CT)<0.01) inMMP-2 and MMP-9, respectively (Lewis B P, et al. Conserved seed pairing,often flanked by adenosines, indicates that thousands of human genes aremicroRNA targets. Cell. 2005; 120(1):15-20; Grimson A, et al. MicroRNAtargeting specificity in mammals: determinants beyond seed pairing. Mol.Cell. 2007; 27(1):91-105). To confirm the initial TargetScan results forMMP-2, a series of other bioinformatics databases were also consulted.Of the 8 additional sites that were examined, a target binding site formiR-29a was identified in the 3′UTR of MMP-2 by 6 of the 8 sitesincluding: RNAhybrid (internet sitebibiserv.techfak.uni-bielefeld.de/rnahybrid/), NBmiRTar (internet sitewotan.wistar.upenn.edu/NBmiRTar/), PicTar (internet sitepictar.bio.nyu.edu/), RNA22 (internet sitecbcsrv.watson.ibm.com/rna22.html), miRanda (web sitemicrorna.org/microrna/home.do), and PITA (internet sitegenie.weizmann.ac.il/). Two sites, DIANA-microT (internet sitediana.pcbi.upenn.edu/cgi-bin/micro_t.cgi), and mirTarget2 (internet sitemirdb.org/miRDB/) failed to identify a potential target binding domain.

v. Viral transduction of vascular smooth muscle cells

Human primary aortic vascular smooth muscle cells (PromoCell,Heidelberg, Germany; Cat#C-12533) were maintained in Smooth Muscle CellGrowth Medium 2 (PromoCell, Cat#C-22062) with 10% heat-inactivated FetalBovine Serum (Cat#16000-044, Invitrogen, Carlsbad, Calif.), 5 μg/mlAmphotericin B (PromoCell, Cat#C-42040) and 0.5 mg/ml Gentamicin(PromoCell, C-42060) at 37° C. in a humidified 5% CO₂ incubator. Thecells (2×10⁵ cells, passage #<8) were seeded into T-25 culture flasks,grown to 70% confluence, and exposed to lentivirus (6.6×10⁶ PFU/ml)encoding either hsa-miR-29a (precursor form; pMIRNAl-hsa-mir-29a,Cat#CS970MR-1, System Biosciences, Mountain View, Calif.), anti-miR-29a(pmiRZip-29a, Cat#CS970MZ-1, System Biosciences), or a non-targetingmismatch control (pGreenPuro Scramble Hairpin Control, Cat#MZIP000-VA-1,System Biosciences) using Transdux reagent (LV850A-1, SystemBiosciences) to improve transduction efficiency, or exposed to Transduxreagent alone (vehicle control). Five days post-transduction the cellswere harvested using enzyme-free Cell Dissociation Buffer(Cat#13151-014, Invitrogen). The cells were centrifuged and resuspendedin cold acidic extraction buffer containing protease inhibitor cocktail(Cat#20-201, Millipore, Billerica, Mass.), prior to analysis by gelatinzymography.

vi. Gelatin Zymography

The relative abundance of MMP-2 and MMP-9 from the aortic tissuespecimens or vascular smooth muscle cells was determined by gelatinzymography (Ikonomidis J S, et al. Expression of matrixmetalloproteinases and endogenous inhibitors within ascending aorticaneurysms of patients with Marfan syndrome. Circulation. 2006; 114(1Suppl):1365-370). The aortic tissue specimens were homogenized in coldacidic extraction buffer using the Qiagen Tissuelyser. Tissue and cellhomogenates were centrifuged (4° C., 10 min, 1200×g) and proteinconcentrations were determined (BCA Protein Assay, Pierce, Rockford,Ill.). Aortic or cellular extracts (10 μg total protein) werefractionated on non-denaturing 10% polyacrylamide gels containing 0.1%(w/v) gelatin (Invitrogen Corporation, Carlsbad, Calif.). The gels werethen equilibrated and incubated in Zymogram Developing Buffer(Invitrogen) for 18 hours at 37° C. After staining with 0.5% CoomassieBrilliant Blue (2 hours, room temperature), the gels were destained toreveal regions of gelatin clearance. The relative abundance of theactive and latent forms of MMP-2 and MMP-9 (as verified by recombinantMMP-2 and MMP-9 standards) were then determined by densitometry usingthe Gel-Pro Analyzer software package (v3.1.14, Media Cybernetics Inc.,Silver Spring, Md.).

vii. Confocal Microscopy

Human primary aortic vascular smooth muscle cells were seeded in 35 mmglass-bottom dishes (Cat#P35GC-1.5-20-C; MatTek Corporation, Ashland,Mass.). When 70% confluency was reached, the cells were transduced withlentiviral constructs, as described above, encoding the hsa-miR-29aprecursor or the hsa-anti-miR-29a, or treated with Transdux reagent(vehicle control) alone. Five-days post-transduction the cells werefixed for 20 min with fresh 3.7% paraformaldehyde. The cells were thenwashed with PBS (6×5 min), and then permeabilized with 0.1% Triton X-100for 10 min. Following a second PBS wash (3×5 min), the cells wereblocked in 10% normal goat serum for 1 hr. Human MMP-2 (active andlatent forms) was then identified by incubating the cells with a rabbitpolyclonal antibody (1:200, Cat#ab37150, Abcam, Cambridge, Mass.) for 2hr in 3% normal goat serum at room temperature. The cells were againwashed with PBS (5×3 min), and then incubated in secondary antibody(1:500, AlexaFluor647 goat-anti-rabbit IgG; Cat#A-21244, InvitrogenCorporation) for 30 min at room temperature in the dark. Each viralvector carries a bicistronic copy of green fluorescent protein (GFP)under control of an independent promoter. Cells were then examined byconfocal microscopy using a Zeiss LSM 510 Meta Confocal Microscope witha Plan-Neofluar 43×/1.3 oil objective. Dual fluorescence (GFP, ex=488nm, em=band-pass 505-530 nm; AlexFluor647, ex=633, em=long pass 650) wasrecorded and the images exported as .tif files.

viii. Data Analysis

To determine miR expression, the relative change in cycle thresholdvalue (ΔCt) from the internal control snRNAU6, was computed. Expressionwas then calculated for each normal and TAA specimen using the equationExpression=2^(−(ΔCt)) which is premised on the fact that each CT valueis in direct proportion to the amount of microRNA present at thebeginning of the reaction. The results were articulated as a percentchange from normal aorta to avoid any bias in total miR concentrationsintroduced by the pre-amplification step. The mean percent change in miRexpression from normal aorta was then calculated. Specimens which didnot cycle, or had Ct values higher than 35, were removed from analysis.Additionally, any expression values that fell more than 2 standarddeviations away from the mean were considered outliers and removed fromanalysis so as not to bias the expression results. Results wereexpressed as mean±SEM.

Relative protein abundance of MMP-2 and MMP-9 were determined fromdensitometry of zymographic gels. Integrated optical densities weredetermined for each specimen and the mean percent change of the ratio ofactive:total MMP from normal aorta was calculated (outliers wereidentified as values falling more than 2 standard deviations away fromthe mean and removed from the data set), and the results were expressedas mean±SEM.

All statistical procedures were carried out using the Stata statisticalpackage (Intercooled Stata v8.2; StataCorp LP, College Station, Tex.).Patient demographics were compared by χ² analysis (gender) or one-wayANOVA (prcompw module) with Tukey's post-hoc analysis (age). Changes inmiR expression and relative protein abundance were determined usingtwo-tailed one-sample mean comparisons versus normal aorta set at 100%,and differences between groups were determined using one-way ANOVA(prcompw module) with Tukey's post-hoc analysis; in both cases values ofp<0.05 were considered significantly different. Differences in miRexpression between the normal and aneurysm groups as determined bymicroarray, were calculated using a Student's t-test. Values of p<0.05were considered to have a significant change in expression.Additionally, differences in miR detectability between the normal andaneurysm groups were calculated using chi-square analysis (tabi command,Stata v8.2). Accordingly, if a given microRNA was detectable in 3 ormore specimens of a single group when compared to no detection in theother group, the miR was considered to have significant differentialdetectability with p<0.05. Relationships between miR expression or MMPprotein abundance and aortic size, as well as the relationship betweenmiR expression and total MMP-2 protein abundance, were determined usinglinear least-squares regression analysis. Results were reportedgraphically and a correlation constant (r-value) and p-value weredetermined (pwcorr and regress modules); relationships with p<0.05 wereconsidered significant.

3. Results

Quantitative real-time polymerase chain reaction (QPCR) was used todetermine the relative expression levels of miRs-1, -21, -29a, -133a,-208, -486-5p, and -760 as compared to snRNAU6, a ubiquitous smallnuclear RNA component of the spliceosome, used as an endogenous control.The QPCR results revealed significantly reduced expression of five ofthe seven miRs examined; miRs-1, -21, -29a, -133a, and -486-5p (FIGS. 28and 34). Expression of miR-760 was unchanged, and the α-myosin heavychain-encoded (myocardial-restricted) miR-208, was not detected in theaortic specimens. To further verify that the loss of miR expression wasdue specifically to reduced miR levels, and not dramatic changes insnRNAU6, a table of Ct values (mean±SEM) for each miR examined isprovided (Table 4).

TABLE 4 Raw Ct values (Mean ± SEM) for each miR examined in this study.Small RNA Control Control Small TAAs Medium TAAs Large TAAs snRNAU6 16.2± 0.6 16.1 ± 0.9 18.8 ± 0.9 16.9 ± 1.0 miR-1 24.0 ± 0.7 25.1 ± 1.7 28.8± 1.8 29.6 ± 1.7 miR-21 18.6 ± 0.9 18.7 ± 1.5 21.8 ± 1.6 21.8 ± 1.7miR-29a 15.2 ± 0.4 17.8 ± 1.7 21.8 ± 1.7 21.9 ± 1.6 miR-133a 18.8 ± 0.420.5 ± 1.3 22.8 ± 1.4 23.2 ± 1.2 miR-486-5p 21.7 ± 0.5 23.6 ± 1.3 24.8 ±1.7 24.7 ± 1.0 miR-760 24.1 ± 0.3 24.5 ± 0.5 26.0 ± 0.7 26.6 ± 0.5

To further examine miR expression in normal and TAA tissues, microarrayanalysis was performed. The results yielded 37 differentially expressedmiRs between normal aorta and aneurysmal aorta, consisting of 4 miRswith increased expression in the TAA group and 33 miRs with decreasedexpression in the TAA group (FIG. 29A). In addition, there were 106 miRsthat were differentially detected either in normal aorta or aneurysm,but not both. These results yielded 54 miRs that had increasedexpression in the aneurysm group, and 52 miRs that had decreasedexpression (FIG. 29B). If a given microRNA was detected in three or morespecimens from either group (normal or aneurysm) it was considered tohave a significant change in expression (chi-square analysis, p<0.05).These data support the changes in miR expression determined byquantitative PCR for miR-1, miR-29a, miR-133a, and miR-486-5p. Functionsfor the 37 differentially expressed miRs were estimated based onpublished manuscripts reported in PubMed (FIG. 35). Accordingly, 27%target proliferation pathways, 16% target growth arrest pathways, 8%target ECM structure/function, 5% (each) target apoptosis, migration,and angiogenesis, 3% (each) target drug resistance, immune response,autophagy, and cell adhesion pathways, and 22% to date have no reportedfunction.

The quantitative PCR results were then stratified based on aorticdiameter of the TAAs at time of surgical resection; small (4.0-5.0 cm),medium (5.1-6.0 cm), or large (6.1-7.5 cm). The miR expression levelswere determined in each size group and articulated as a percent changefrom miR expression in normal aorta. Interestingly, each of the miRsdetected demonstrated size-dependent changes in expression (FIG. 30A).

Using linear least-squares regression modeling, miR expression wascompared to aortic size for each of the TAA specimens. Severalsignificant inverse relationships were identified; miR-1 (r=−0.5433,p=0.0109, n=21), miR-21 (r=−0.4132, p=0.0359, n=26), miR-29a (r=−0.5364,p=0.0039, n=27), and miR-133a (r=−0.4247, p=0.0344, n=25) (FIG. 30B).

Potential biological targets were searched using the TargetScanHumandatabase for the presence of conserved nucleotide sequences (7 to 8nucleotides in length), that significantly matched with the seed regions(nucleotides 2-7) of miRs-1, -21, -29a, and -133a.³ Potential bindingsites for miR-29a (conserved, 7mer-8; context score=−0.01, P_(CT)=0.82)and miR-133a (low species conservation, 7mer-8; context score=−0.04,P_(CT)<0.01) were identified in MMP-2 and MMP-9, respectively (FIG. 31A)(Lewis B P, et al. Conserved seed pairing, often flanked by adenosines,indicates that thousands of human genes are microRNA targets. Cell.2005; 120(1):15-20; Friedman R C, et al. Most mammalian mRNAs areconserved targets of microRNAs. Genome Res. 2009; 19(1):92-105; GrimsonA, et al. MicroRNA targeting specificity in mammals: determinants beyondseed pairing. Mol. Cell. 2007; 27(1):91-105).

In order to investigate the relationship between MMP abundance and miRexpression, the relative abundance of MMP-2 and MMP-9 protein levels wasexamined in the same clinical TAA specimens using zymography. Bothactive and latent bands were identified by recombinant standards andtotal MMP was calculated (sum of active+latent), and the percent changein ratio of active:total forms as compared to normal aorta werestratified by aneurysm size; providing both a measure of MMP abundanceand activational state. The data revealed an increased MMP-2active:total ratio in each of the aneurysm size groups, while nodifference in the MMP-9 ratio was observed (FIG. 31B,C). Usinglinear-least squares regression modeling, the increase in active:totalMMP-2 ratio displayed a significant positive relationship to aneurysmsize (r=0.4056, p=0.0262, n=30) indicating that the abundance andactivation of MMP-2 increase with increasing aortic diameter.

Accordingly, to examine the relationship between MMP-2 abundance and miRexpression, human primary aortic vascular smooth muscle cells weretransduced with lentiviral constructs containing a bicistronic copy ofgreen fluorescent protein (GFP) and were designed to overexpress miR29aprecursor or the miR antagonist, anti-miR-29a. Transduced cells wereexamined by confocal microscopy and gelatin zymography for changes inthe abundance of latent and active forms of the MMP-2 protein. Greenfluorescent protein (GFP), driven from an independent promoter on eachviral construct was used to identify transduced cells. The cells werestained with an antibody for total MMP-2, and dual fluorescence wasrecorded. As shown in the transduction vehicle controls, MMP-2 islocalized to the cell periphery in proximity to the plasma membranesurface. In miR-29a transduced cells, overexpression of miR-29a resultedin the attenuation of total MMP-2 staining Conversely, in cellstransduced with anti-miR-29a, overexpression of the miR antagonistresulted in enhanced MMP-2 staining in the peri-nuclear region, as wellas at the periphery of the cell. In order to quantitate the effects ofmodulating miR-29a on the abundance of total MMP-2 protein levels, cellswere transduced with miR-29a precursor or anti-miR-29a and harvested forgelatin zymography (FIG. 32). The data demonstrated that overexpressionof the miR-29a precursor resulted in decreased total MMP-2 abundance.Conversely, overexpression of anti-miR-29a resulted in enhanced totalMMP-2 protein abundance. Additionally, when vascular smooth muscle cellswere transduced with a non-targeting mismatch control lentivirus, nochange in total MMP-2 protein abundance was observed (FIG. 36).

Lastly, to examine the relationship between total MMP-2 proteinabundance and miR-29a expression in the aortic specimens, linearleast-squares regression modeling was again performed, and a significantinverse relationship between miR-29a expression and total MMP-2abundance was identified (r=−0.4198, p=0.0209, n=30); indicating that asmiR-29a decreased, the relative abundance of MMP-2 increased (FIG. 33).

4. Discussion

Thoracic aortic aneurysm disease results as a consequence ofpathological remodeling within the aortic vascular ECM. This remodelingprocess induces a progressive weakening of the ECM through a variety ofmechanisms including altered collagen deposition/processing, and theelevation of proteinase activity (Damodarasamy M, et al. CollagenExtracts Derived From Young and Aged Mice Demonstrate DifferentStructural Properties and Cellular Effects in Three-Dimensional Gels. JGerontol A Biol Sci Med. Sci. 2010; 65(3):209-218; Lakatta EG, Levy D.Arterial and cardiac aging: major shareholders in cardiovascular diseaseenterprises: Part II: the aging heart in health: links to heart disease.Circulation. 2003; 107(2):346-354; Lakatta EG, Levy D. Arterial andcardiac aging: major shareholders in cardiovascular disease enterprises:Part I: aging arteries: a “set up” for vascular disease. Circulation.2003; 107(1):139-146). These changes within the aortic wall result indecreased compliance and competence, culminating in aortic dilatationand eventual rupture. In an effort to further understand the molecularpathogenesis of TAA development, this study examined the expression of afocused set of miRs in clinical TAA specimens compared to aorticexpression levels in patients without aneurysm disease. It washypothesized that TAA development would coincide with alterations inspecific miR expression that could affect the induction of targetproteins which contribute to aortic vascular remodeling. This studyfocused on the expression of seven miRs that were selected because oftheir reported involvement in the regulation of specific protein targetswithin the cardiovascular system; including several MMPs and ECMcomponents (collagens, elastin, and microfibrillar proteins) (Small E M,et al. MicroRNAs add a new dimension to cardiovascular disease.Circulation. 2010; 121(8):1022-1032). The unique findings of this studywere four-fold.

First, within the focused set of miRs studied, five were found to havedecreased expression in clinical ascending TAA specimens as compared tonormal aorta (miRs-1, -21, -29a, -133a, and -486) as established byquantitative PCR. MicroRNA expression values were calculated fromnormalized C_(T) values (ΔC_(T)) using the equation (expression2^(−ΔCT)) for each specimen, premised on the fact that each C_(T) valueis in direct proportion to the amount of microRNA present at thebeginning of the reaction. While factors such as PCR amplificationefficiencies can influence the amount of product generated, and therebyinfluence the sensitivity of an individual reaction, the distribution ofmeasured miR values within the sampled cohort for this study (n=10normal, and n=30 TAA specimens) provided sufficient power todiscriminate a 50%-80% reduction in miR concentration between the normaland TAA groups (corresponding to a C_(T) value increase of 1 to 1.2cycles). The statistical power for comparing miR values between the twogroups was greater than 0.92 for all miRs measured except miR-760, wherethe power was 0.29. Based on expectations of structural remodelingwithin the aortic wall in the aneurysm specimens, it was anticipatedthat the protein levels of multiple MMPs and ECM proteins would beinduced in response to TAA development (Jones J A, et al. Alterations inmembrane type-1 matrix metalloproteinase abundance after the inductionof thoracic aortic aneurysm in a murine model. Am J Physiol Heart CircPhysiol. 2010; 299(1):H114-124). Because miRs function to degrade mRNAor repress message translation, it was not a surprise to observe asignificant loss of miR expression, knowing that many of the putativetargets would increase in response to the disease state. This loss ofmiR expression in vascular disease is consistent with other reportsexamining miR expression by microarray. For example, Liao and coworkersprofiled miR expression in a cohort of ascending aortic specimens bymicroarray, comparing normal patients to those with aortic dissection.They observed 74 miRs that were differentially expressed with apredominance of miRs (n=56) showing reduced expression in the dissectedtissue (Liao M, et al. A microRNA profile comparison between thoracicaortic dissection and normal thoracic aorta indicates the potential roleof microRNAs in contributing to thoracic aortic dissection pathogenesis.J Vasc Surg. 2011; doi:10.1016/j.jvs.2010.11.113).

To further examine miR expression differences between the aneurysmal andnormal aorta, microarray analysis was performed, and similar to Liao andcoworkers, of the 37 differentially expressed miRs identified, 33 werefound to have decreased expression. Included in the group of 33 miRswith decreased expression, were the miR-143/miR-145 cluster, previouslyreported by Elia and colleagues to be decreased in human ascendingaortic aneurysm biopsies (Elia L, et al. The knockout of miR-143 and-145 alters smooth muscle cell maintenance and vascular homeostasis inmice: correlates with human disease. Cell Death Differ. 2009;16(12):1590-1598), and miR-29a, identified in this study as havingsignificantly decreased expression in TAA specimens. Based on previousliterature, the majority of these differentially expressed miRs targetpathways involved in proliferation, growth arrest, and ECMstructure/function. Taken together, this study indicates that the lossof specific miR expression can allow for the elaboration of specificprotein targets that contribute to the aortic remodeling process duringTAA development.

Second, several miRs displayed a significant relationship between theloss miR expression and the enhancement of aortic diameter (miRs-1, -21,-29a, and -133a). These results indicated that dynamic changes in miRexpression levels likely occur during aneurysm progression, andemphasize that the loss of ongoing translational repression can play akey role in modulating the determinants of vascular remodeling.

Third, because alterations in miR expression have been shown to modulatetarget protein abundance, a bioinformatics approach was used to identifyputative miR target sequences in genes known to be involved in TAAformation and progression. As a first approach, the TargetScanHumandatabase was queried with the sequence of full-length transcripts forseveral MMPs and ECM structural proteins. Potential biological targetswere screened for the presence of conserved nucleotide sequences thatsignificantly matched the seed regions of miRs-1, -21, -29a, and -133a(Lewis B P, et al. Conserved seed pairing, often flanked by adenosines,indicates that thousands of human genes are microRNA targets. Cell.2005; 120(1):15-20). Results identified a highly conserved miR-29atarget sequence (7mer-8) in the 3′ UTR of MMP-2, and a less wellconserved miR-133a target sequence (7mer-8) in the 3′ UTR of MMP-9. Workby Bartel and colleagues, has indicated that conserved 7mer-8 targetsequences are significant predictors for high efficacy miR-mediatedmessage destabilization (Bartel D P. MicroRNAs: target recognition andregulatory functions. Cell. 2009; 136(2):215-233). The MMPs are adiverse family of proteases capable of degrading all components of thevascular ECM. In numerous studies using human specimens and animalmodels, increased protein levels of MMP-2 and MMP-9 have been directlyimplicated in aneurysm development in both the abdominal and thoracicaorta (Ikonomidis J S, et al. Expression of matrix metalloproteinasesand endogenous inhibitors within ascending aortic aneurysms of patientswith Marfan syndrome. Circulation. 2006; 114(1 Suppl):1365-370; Absi TS, et al. Altered patterns of gene expression distinguishing ascendingaortic aneurysms from abdominal aortic aneurysms: complementary DNAexpression profiling in the molecular characterization of aorticdisease. J Thorac Cardiovasc Surg. 2003; 126(2):344-357; discission 357;Boyum J, et al. Matrix metalloproteinase activity in thoracic aorticaneurysms associated with bicuspid and tricuspid aortic valves. J ThoracCardiovasc Surg. 2004; 127(3):686-691; Ikonomidis J S, et al. Effects ofdeletion of the matrix metalloproteinase 9 gene on development of murinethoracic aortic aneurysms. Circulation. 2005; 112(9 Suppl):1242-248;Ikonomidis J S, et al. Expression of matrix metalloproteinases andendogenous inhibitors within ascending aortic aneurysms of patients withbicuspid or tricuspid aortic valves. J Thorac Cardiovasc Surg. 2007;133(4):1028-1036; Jones J A, et al. Spatiotemporal expression andlocalization of matrix metalloproteinase-9 in a murine model of thoracicaortic aneurysm. J Vasc Surg. 2006; 44(6):1314-1321; Longo G M, et al.Matrix metalloproteinases 2 and 9 work in concert to produce aorticaneurysms. J Clin Invest. 2002; 110(5):625-632; McMillan W D, et al. Insitu localization and quantification of seventy-two-kilodalton type IVcollagenase in aneurysmal, occlusive, and normal aorta. J Vasc Surg.1995; 22(3):295-305; McMillan W D, et al. In situ localization andquantification of mRNA for 92-kD type IV collagenase and its inhibitorin aneurysmal, occlusive, and normal aorta. Arterioscler Thromb VascBiol. 1995; 15(8):1139-1144; Tamarina N A, et al. Expression of matrixmetalloproteinases and their inhibitors in aneurysms and normal aorta.Surgery. 1997; 122(2):264-271; discussion 271-262). Therefore, thepresence of these predicted target sequences indicates that theseproteases can be subject to post-transcriptional or translationalregulation by miR expression (Bartel D P. MicroRNAs: target recognitionand regulatory functions. Cell. 2009; 136(2):215-233; Liu Y, et al.Renal medullary microRNAs in Dahl salt-sensitive rats: miR-29b regulatesseveral collagens and related genes. Hypertension. 2010; 55(4):974-982;Steele R, et al. MBP-1 upregulates miR-29b that represses Mc1-1,collagens, and matrix-metalloproteinase-2 in prostate cancer cells.Genes Cancer. 2010; 1(4):381-387). The protein levels of MMP-2 and MMP-9were therefore examined in the clinical TAA specimens, and the indicesof MMP-2 abundance were found to be elevated compared to normal aorticspecimens. Moreover, a positive relationship was identified between theactive:total MMP-2 ratio and aortic diameter, indicating MMP-2 as animportant mediator of aneurysm formation, consistent with previousfindings (Jones J A, et al. Alterations in membrane type-1 matrixmetalloproteinase abundance after the induction of thoracic aorticaneurysm in a murine model. Am J Physiol Heart Circ Physiol. 2010;299(1):H114-124; Ikonomidis J S, et al. Expression of matrixmetalloproteinases and endogenous inhibitors within ascending aorticaneurysms of patients with bicuspid or tricuspid aortic valves. J ThoracCardiovasc Surg. 2007; 133(4):1028-1036; Longo G M, et al. Matrixmetalloproteinases 2 and 9 work in concert to produce aortic aneurysms.J Clin Invest. 2002; 110(5):625-632; Borges L F, et al. Tissue diffusionand retention of metalloproteinases in ascending aortic aneurysms anddissections. Human pathology. 2009; 40(3):306-313; Chung A W, et al.Loss of elastic fiber integrity and reduction of vascular smooth musclecontraction resulting from the upregulated activities of matrixmetalloproteinase-2 and -9 in the thoracic aortic aneurysm in Marfansyndrome. Circ Res. 2007; 101(5):512-522; LeMaire S A, et al. Matrixmetalloproteinases in ascending aortic aneurysms: bicuspid versustrileaflet aortic valves. J Surg Res. 2005; 123(1):40-48; Sinha I, etal. A biologic basis for asymmetric growth in descending thoracic aorticaneurysms: a role for matrix metalloproteinase 9 and 2. J Vasc Surg.2006; 43(2):342-348).

In order to demonstrate a direct relationship between miR-29a and MMP-2,lentiviral vectors were used to transduce human primary aortic vascularsmooth muscle cells with either the miR-29a precursor, or ananti-miR-29a, designed to knockdown cellular miR-29a levels. Thetransduced cells were then examined for changes in MMP-2 protein levelsby confocal microscopy and gelatin zymography. The data demonstratedoverexpression of miR-29a resulted in the attenuation of MMP-2 proteinlevels, while the overexpression of anti-miR-29a resulted in enhancedMMP-2 protein abundance. These results identify MMP-2 as one of thetarget proteins for miR-29a, and support previous reports that haveidentified miR-29 as a transcriptional regulator of MMP-2 (Liu Y, et al.Renal medullary microRNAs in Dahl salt-sensitive rats: miR-29b regulatesseveral collagens and related genes. Hypertension. 2010; 55(4):974-982;Steele R, Mott J L, Ray R B. MBP-1 upregulates miR-29b that repressesMc1-1, collagens, and matrix-metalloproteinase-2 in prostate cancercells. Genes Cancer. 2010; 1(4):381-387).

Last, in order to further implicate a role for miR-29a in aneurysmdevelopment, regression modeling was performed and a significant inverserelationship between miR-29a expression and total MMP-2 proteinabundance was demonstrated. This unique observation indicated that theloss of aortic miR-29a levels can permit the elaboration of MMP-2translation within the developing aneurysm, and can identify a potentialmechanism by which MMP-2 protein induction occurs during TAAdevelopment.

While the present study has identified miR-29a as a potential regulatorof MMP-2 in the aorta, it is important to note that each miR can targetupwards of 200 different mRNAs, many of which may impact TAAdevelopment. For example, in silico mapping also identified miR-29atarget binding sites in type-I and type-III collagens, elastin, andfibrillin-1. Dysregulation of any of these putative targets may bedeleterious to the structure and function of the thoracic aorta. Thus,altered miR expression and function within clinical TAAs may play aprincipal role in aneurysm progression by fine-tuning the proteinabundance of a cassette of specific genes that together can influence atissue-specific response.

The unique results of this study carry several significant clinicalimplications. First, miR expression profiles can provide significantinsight into the identification of potential upstream mediators ofaortic ECM remodeling and can reveal therapeutic strategies for thetreatment of TAA disease. Second, the altered miR expression profilesidentified in this study can be expanded to lay the foundation for thedevelopment of diagnostic or prognostic bioassays with the potential todefine phases of disease progression, informing of the best time tointervene surgically or even indicating the potential for aorticrupture. Lastly, understanding the regulation of specific miR expressionin relation to the pathophysiology behind TAA development, can alsoprovide novel therapeutic strategies aimed at modulating miR expressionin order to arrest aneurysm development or even reverse dilatation.

Taken together, this study identified that the loss of miR expressionduring clinical TAA development can play a key role in exacerbatingpathological remodeling by removing an inhibitory signal that normallyattenuates MMP production. Importantly, these data indicate that thesemiRs have biological and clinical relevance to the behavior of TAAs, andcan provide significant targets for diagnostic and therapeuticapplications.

REFERENCES

-   Chen X, Ba Y, Ma L, Cai X, Yin Y, Wang K, Guo J, Zhang Y, Chen J,    Guo X, Qibin L, Li X, Wang W, Zhang Y, Wang J, Jiang X, Xiang Y, Xu    C, Zheng P, Zhang J, Li R, Zhang H, Shang X, Gong T, Ning G, Wang J,    Zen K, Zhang J, Zhang C. Characterization of microRNAs in serum: a    novel class of biomarkers for diagnosis of cancer and other    diseases. Cell Research. 2008; 18:997-1006.-   Divakaran V, Mann D L. The Emerging Role of MicroRNAs in Cardiac    Remodeling and Heart Failure. Circ Res. 2008; 103:1072-1083.-   Dong S, Cheng Y, Yang J, Li J, Liu X, Wang X, Wang D, Krall T J,    Delphin E S, Zhang C. MicroRNA Expression Signature and the Role of    MicroRNA-21 in the Early Phase of Acute Myocardial Infarction. J    Biol. Chem. 2009; 284(43):29514-25.-   Duisters R F, Tijsen A J, Schroen B, Leenders J J, Lentink V, van    der Made I, Herias V, van Leeuwen R E, Schellings M W, Barenbrug P,    Maessen J G, Heymans S, Pinto Y M, Creemers E E. miR-133 and miR-30    Regulate Connective Tissue Growth Factor. Implications for a Role of    MicroRNAs in Myocardial Matrix Remodeling. Circ Res. 2009;    104:170-178.-   Li J, Yao B, Huang H, Wang Z, Sun C, Fan Y, Chang Q, Li S, Wang X,    Xi J. Real-Time Polymerase Chain Reaction MicroRNA Detection Based    on Enzymatic Stem-Loop Probes Ligation. Anal Chem. 2009 Jul. 1;    81(13):5446-51.-   Liu N, Bezprozvannaya S, Williams A H, Qi X, Richardson J A,    Bassel-Duby R, Olson E N. microRNA-133a regulates cardiomyocyte    proliferation and suppresses smooth muscle gene expression in the    heart. Genes Dev. 2008; 22(23):3242-54.-   Mitchell P S, Parkin R K, Kroh E V, Fritz B R, Wyman S K,    Pogosova-Agadjanyan E L, Peterson A, Noteboom J, O'Briant K C, Allen    A, Lin D W, Urban N, Drescher C W, Knudsen B S, Stirewalt D L,    Gentleman R, Vessella R L, Nelson P S, Martin D B, Tewari M.    Circulating microRNAs as stable blood-based markers for cancer    detection. Proc Natl Acad Sci USA. 2008; 105 (30): 10513-8.-   Roy S, Khanna S, Hussain S A, Biswas S, Azad A, Rink C, Gnyawali S,    Shilo S, Nuovo G J, Sen C K. MicroRNA expression in response to    murine myocardial infarction: miR-21 regulates fibroblast    metalloprotease-2 via phosphatase and tensin homologue. Cardiovasc    Res. 2009; 82(1):21-9.-   Small E M, Frost R J A, Olson E N. MicroRNAs Add a New Dimension to    Cardiovascular Disease. Circulation. 2010; 121:1022-1032.-   van Rooij E, Sutherland L B, Thatcher J E, DiMaio M, Naseem R H,    Marshall W S, Hill J A, Olson E N. Dysregulation of microRNAs after    myocardial infarction reveals a role of miR-29 in cardiac fibrosis.    Proc Natl Acad. Sci. 2008; 105(35):13027-32.

It is understood that the disclosed method and compositions are notlimited to the particular methodology, protocols, and reagents describedas these may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present invention which willbe limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “amicroRNA” includes a plurality of such microRNAs, reference to “themicroRNA” is a reference to one or more microRNAs and equivalentsthereof known to those skilled in the art, and so forth.

“Optional” or “optionally” means that the subsequently described event,circumstance, or material may or may not occur or be present, and thatthe description includes instances where the event, circumstance, ormaterial occurs or is present and instances where it does not occur oris not present.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, also specifically contemplated and considered disclosed isthe range from the one particular value and/or to the other particularvalue unless the context specifically indicates otherwise. Similarly,when values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms another,specifically contemplated embodiment that should be considered disclosedunless the context specifically indicates otherwise. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint unless the context specifically indicates otherwise. Finally,it should be understood that all of the individual values and sub-rangesof values contained within an explicitly disclosed range are alsospecifically contemplated and should be considered disclosed unless thecontext specifically indicates otherwise. The foregoing appliesregardless of whether in particular cases some or all of theseembodiments are explicitly disclosed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present method andcompositions, the particularly useful methods, devices, and materialsare as described. Publications cited herein and the material for whichthey are cited are hereby specifically incorporated by reference.Nothing herein is to be construed as an admission that the presentinvention is not entitled to antedate such disclosure by virtue of priorinvention. No admission is made that any reference constitutes priorart. The discussion of references states what their authors assert, andapplicants reserve the right to challenge the accuracy and pertinency ofthe cited documents. It will be clearly understood that, although anumber of publications are referred to herein, such reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in the art.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers or steps.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the method and compositions described herein. Suchequivalents are intended to be encompassed by the following claims.

1. A method comprising detecting one or more target microRNAs in tissueor a body fluid of a subject, wherein the level or amount of the one ormore target microRNAs indicates the risk, presence, severity, or acombination, of thoracic aortic aneurysm (TAA) in the subject, ofdiastolic heart failure in the subject, of left ventricular hypertrophyin the absence of diastolic heart failure in the subject, of leftventricular remodeling in the subject, that the subject experiencedischemia-reperfusion, or a combination.
 2. The method of claim 1,wherein the one or more target microRNAs comprises miR-1, miR-21,miR-29a, miR-133a, and miR-486-5p, or a combination, wherein a level oramount of miR-1, miR-21, miR-29a, miR-133a, and miR-486-5p, or acombination, less than the level or amount in control subjects,reference subjects, normal subjects, or a combination, indicates therisk, presence, severity, or a combination, of TAA in the subject. 3.The method of claim 1, wherein the one or more target microRNAscomprises miR-455-3p, miR-1268, miR-338-5p, miR-940, miR-1323,miR-768-3p, miR-574-3p, miR-106b, miR-451, miR-100, miR-125b, miR-195,miR-19b, miR-30d, miR-15b, miR-125a-5p, miR-143, miR-193b, miR-16,miR-27a, miR-29a, miR-30a, miR-27b, miR-92a, miR-140-5p, let-7i,miR-151-5p, miR-140-3p, miR-24, miR-23a, miR-145, miR-199b-3p,miR-199a-3p, miR-361-5p, miR-130a, miR-22, and miR-497, wherein a levelor amount, for a threshold percentage or more of the target microRNAsmiR-455-3p, miR-1268, miR-338-5p, miR-940, miR-1323, miR-768-3p,miR-574-3p, miR-106b, miR-451, miR-100, miR-125b, miR-195, miR-19b,miR-30d, miR-15b, miR-125a-5p, miR-143, miR-193b, miR-16, miR-27a,miR-29a, miR-30a, miR-27b, miR-92a, miR-140-5p, let-7i, miR-151-5p,miR-140-3p, miR-24, miR-23a, miR-145, miR-199b-3p, miR-199a-3p,miR-361-5p, miR-130a, miR-22, and miR-497: (a) of less than the level oramount in control subjects, reference subjects, normal subjects, or acombination, for miR-1323, miR-768-3p, miR-574-3p, miR-106b, miR-451,miR-100, miR-125b, miR-195, miR-19b, miR-30d, miR-15b, miR-125a-5p,miR-143, miR-193b, miR-16, miR-27a, miR-29a, miR-30a, miR-27b, miR-92a,miR-140-5p, let-7i, miR-151-5p, miR-140-3p, miR-24, miR-23a, miR-145,miR-199b-3p, miR-199a-3p, miR-361-5p, miR-130a, miR-22, and miR-497, and(b) of greater than the level or amount in control subjects, referencesubjects, normal subjects, or a combination, for miR-455-3p, miR-1268,miR-338-5p, and miR-940, indicates the risk, presence, severity, or acombination, of thoracic aortic ancurysmTAA in the subject.
 4. Themethod of claim 3, wherein the threshold percentage is 60%.
 5. Themethod of claim 3, wherein the threshold percentage is 80%.
 6. Themethod of claim 3, wherein the threshold percentage is 90%.
 7. Themethod of claim 1, wherein the one or more target microRNAs comprisesmiR-1, miR-21, miR-29a, and miR-133a, or a combination, wherein thelower the level or amount of miR-1, miR-21, miR-29a, miR-133a, andmiR-486-5p, or a combination, less than the level or amount in controlsubjects, reference subjects, normal subjects, or a combination, thelarger the TAA in the subject.
 8. The method of claim 1, wherein thelower the level or amount of miR-29a less than the level or amount incontrol subjects, reference subjects, normal subjects, or a combination,the higher the level of MMP-2 in the subject.
 9. The method of claim 1,wherein the lower the level or amount of miR-133a less than the level oramount in control subjects, reference subjects, normal subjects, or acombination, the higher the level of MMP-9 in the subject.
 10. Themethod of claim 1, wherein prior to detection, the subject is suspectedof having TAA.
 11. The method of claim 1, wherein the subject has leftventricular hypertrophy, wherein the one or more target microRNAscomprises miR-1, miR-21, miR-29a, miR-133a, and miR-760, or acombination, wherein a decrease in the level or amount of miR-1, miR-21,miR-29a, miR-133a, and miR-760, or a combination, from a level or amountgreater than the level or amount in control subjects, referencesubjects, normal subjects, or a combination, indicates the risk,development, presence, severity, or a combination, of diastolic heartfailure in the subject.
 12. The method of claim 1, wherein the subjecthas left ventricular hypertrophy, wherein the one or more targetmicroRNAs comprises miR-1, miR-21, miR-29a, miR-133a, and miR-760, or acombination, wherein a decrease in the level or amount of miR-1, miR-21,miR-29a, miR-133a, and miR-760, or a combination, from a level or amountin subjects with left ventricular hypertrophy but not diastolic heartfailure, indicates the risk, development, presence, severity, or acombination, of diastolic heart failure in the subject.
 13. The methodof claim 11, wherein the decrease in the level or amount of the one ormore target microRNAs is determined by detecting the level or amount ofthe one or more target microRNAs at different times.
 14. The method ofclaim 13, wherein the level or amount of the one or more targetmicroRNAs at one or more of the different times is greater than thelevel or amount in control subjects, reference subjects, normalsubjects, or a combination.
 15. The method of claim 1, wherein thesubject has left ventricular hypertrophy, wherein the one or more targetmicroRNAs comprises miR-1, miR-21, miR-29a, miR-133a, and miR-760, or acombination, wherein a level or amount of miR-1, miR-21, miR-29a,miR-133a, and miR-760, or a combination, about the same as the level oramount in control subjects, reference subjects, normal subjects, or acombination, indicates the presence of diastolic heart failure in thesubject.
 16. The method of claim 1, wherein the subject has leftventricular hypertrophy, wherein the one or more target microRNAscomprises miR-1, miR-21, miR-29a, miR-133a, and miR-760, or acombination, wherein a level or amount of miR-1, miR-21, miR-29a,miR-133a, and miR-760, or a combination, greater than the level oramount in control subjects, reference subjects, normal subjects, or acombination, indicates the absence of diastolic heart failure in thesubject.
 17. The method of claim 1, wherein the one or more targetmicroRNAs comprises miR-1, miR-133a, miR-760, or a combination, whereina level or amount of miR-1, miR-133a, miR-760, or a combination, greaterthan the level or amount in control subjects, reference subjects, normalsubjects, or a combination, indicates that the subject experiencedischemia-reperfusion.
 18. The method of claim 1, wherein prior todetection, the subject is suspected of having experiencedischemia-reperfusion.
 19. The method of claim 1, wherein the one or moretarget microRNAs are detected following myocardial infarction.
 20. Themethod of claim 19, wherein the one or more target microRNAs comprisesmiR-1, miR-21, or a combination, wherein the one or more targetmicroRNAs are detected on or about 2 days following myocardialinfarction, wherein a level or amount of miR-1, miR-21, or acombination, less than the level or amount in control subjects,reference subjects, normal subjects, or a combination, indicates ahigher risk of left ventricular remodeling in the subject.
 21. Themethod of claim 19, wherein the one or more target microRNAs comprisesmiR-29a, wherein the one or more target microRNAs are detected on,about, or between 4 and 10 days following myocardial infarction, whereina level or amount of miR-29a more than the level or amount in controlsubjects, reference subjects, normal subjects, or a combination,indicates a higher risk of increased left ventricular end diastolicvolume developing in the subject.
 22. The method of claim 21, whereinthe one or more target microRNAs are detected on, about, or between 5and 8 days following myocardial infarction.
 23. The method of claim 21,wherein the one or more target microRNAs are detected on or about 5 daysfollowing myocardial infarction.
 24. The method of claim 1, wherein theone or more target microRNAs are detected in a tissue.
 25. The method ofclaim 24, wherein the tissue is cardiac tissue.
 26. The method of claim24, wherein the tissue is thoracic aortic tissue.
 27. The method ofclaim 1, wherein the one or more target microRNAs are detected in a bodyfluid.
 28. The method of claim 27, wherein the body fluid is plasma. 29.The method of claim 1, wherein the one or more target microRNAs aredetected in a tissue sample or a body fluid sample collected from thesubject.
 30. The method of claim 1, wherein detection of the one or moremicroRNAs is accomplished by altering the tissue or body fluid so thatthe microRNAs produce a signal.
 31. The method of claim 1, wherein thealteration of the tissue or body sample comprises amplifying themicroRNAs.
 32. The method of claim 1, wherein the level of the one ormore target microRNAs comprises the measured level of the one or moretarget microRNAs normalized to the measured level of a reference RNA inthe tissue or body fluid.
 33. The method of claim 32, wherein thereference RNA is snRNA U6.
 34. The method of claim 1, wherein the levelof the one or more target microRNAs comprises the measured level of theone or more target microRNAs expressed as the fold difference of themeasured level of the one or more target microRNAs to the measured levelof the one or more target microRNAs in a reference subject.
 35. Themethod of claim 1, wherein the level of the one or more target microRNAscomprises the measured level of the one or more target microRNAsnormalized to the measured level of a reference RNA in the tissue orbody fluid expressed as the fold difference of the normalized level ofthe one or more target microRNAs to the measured level of the one ormore target microRNAs in the same tissue or body fluid of referencesubject normalized to the measured level of a reference RNA in thetissue or body fluid of the reference subject.
 36. The method of claim34, wherein the level of the one or more target microRNAs in a referencesubject is measured at the same time as the level of the one or moretarget microRNAs is measured in the subject.
 37. The method of claim 34,wherein the level of the one or more target microRNAs in a referencesubject is measured at a different time than the level of the one ormore target microRNAs is measured in the subject.
 38. The method ofclaim 34, wherein the level of the one or more target microRNAs in areference subject is a reference level.
 39. The method of claim 1further comprising treating the subject for thoracic aortic ancurysmTAA.40. The method of claim 1 further comprising treating the subject fordiastolic heart failure.
 41. The method of claim 1 further comprisingtreating the subject for ischemia-reperfusion injury.
 42. The method ofclaim 1 further comprising treating the subject for left ventricularremodeling.